CN115605597A

CN115605597A - Method for generating constitutive bacterial promoters conferring low to moderate expression

Info

Publication number: CN115605597A
Application number: CN202180017085.2A
Authority: CN
Inventors: M·F·菲勒; C·索尔; N·韦尔奇; M·阿佩尔鲍姆; T·施韦德尔
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2020-03-04
Filing date: 2021-03-01
Publication date: 2023-01-13
Also published as: US20230212593A1; KR20220150328A; WO2021175759A1; EP4114954A1

Abstract

The present invention is in the field of molecular biology and provides methods for producing constitutive promoters with low to moderate expression in bacteria and promoters produced by said methods.

Description

Method for generating constitutive bacterial promoters conferring low to moderate expression

Summary of The Invention

Introduction to the invention

Microorganisms are now widely used in industry by taking advantage of their fermentative capabilities. Microorganisms are used in particular as hosts for the fermentative production of various substances, such as enzymes, proteins, chemicals, sugars and polymers. For these purposes, microorganisms are the subject of genetic engineering, aimed at modifying their gene expression in response to the needs of a particular production process. Rational genetic engineering of microorganisms requires target-specific genome editing techniques such as introduction of point mutations, gene deletions, gene insertions, gene duplications.

Many different genome editing methods have been developed for several species. Most of them require the introduction of a double-stranded DNA break or two adjacent single-stranded DNA breaks, to introduce random mutations at specific sites in the genome by non-homologous end joining (NHEJ) or to introduce, replace or delete DNA using homologous recombination repair mechanisms (HR) which require delivery of a donor DNA molecule. The techniques used are, for example, zn-finger nucleases, TALENs, homing endonucleases, etc. Recent developments in CRISPR (clustered regularly interspaced short palindromic repeats) based systems have made genome editing even more attractive due to its precision, efficiency and speed.

The CRISPR system was originally identified as an adaptive defense mechanism for Streptococcus (Streptococcus) bacteria (WO 2007/025097). Those bacterial CRISPR systems rely on guide RNAs (grnas) complexed with splicing proteins to direct degradation of complementary sequences present within the DNA of invasive viruses. The first identified protein, cas9, in the CRISPR/Cas system is a large, monomeric DNA nuclease that is guided by a complex of two non-coding RNAs (crRNA and trans-activating crRNA (tracrRNA)) to a DNA target sequence adjacent to a PAM (protospacer adjacent motif) sequence motif. Later, synthetic RNA chimeras (single guide RNAs or sgrnas) created by fusion of crRNA to tracrRNA were shown to be equally functional (Jinek, m., cylinski, k., fonfara, i., hauer, m., doudna, j.a. and charpienter, e.a. programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity) Science 337 (6096), 816-821.17-8-2012).

Several research groups have found that CRISPR cleavage properties can be used to destroy genes in the genome of almost any organism with unprecedented ease (Mali P et al (2013) science.339 (6121): 819-823. Recently, it has become clear that providing templates for repair allows editing of genomes at almost any site with almost any sequence of interest, thereby converting CRISPRs into powerful gene editing tools (WO/2014/150624, WO/2014/204728).

A key element that drives gene expression in a host cell is the promoter sequence. In order for gene expression to occur, the RNA polymerase must bind to promoter sequences in the vicinity of the gene. Thus, the promoter contains specific DNA sequences that provide binding sites for RNA polymerase and other proteins (i.e., transcription factors) that also recruit RNA polymerase to the recognition sequence. In bacteria, promoters are usually recognized by RNA polymerase and related sigma factors, which are directed to the promoter DNA by binding of activator proteins to their nearby self-DNA binding sites (Lee, d.j., minchi, s.d. and Busby, s.j. activating transcription in bacteria, annu. Rev. Microbiol.66, 125-152.2012). For example, constitutive promoters driving the expression of many housekeeping genes are not associated with activation or derepression by activator or repressor proteins and RNA polymerase binds to the constitutive promoters by the recognition of the sigA-specific DNA sequence elements-35-box and-10-box related sigma factor sigA (also referred to as sig70 in E.coli). The sigA-dependent promoters of Bacillus (Bacillus) and E.coli have been well studied and comparison of the consensus motifs of the sigA promoter sequences suggests that Bacillus-derived and E.coli-derived sigA promoters are cross-recognized by E.coli RNA polymerase and Bacillus RNA polymerase, respectively, and the corresponding sig70 and sigA factors (Helmann, J.D. compatibility and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. (Bacillus subtilis) σ A-dependent promoter sequence codification and analysis: evidence of extended contact between RNA polymerase and upstream promoter DNA. Closed Acids Res.23 (13), 2351-2360.11-327-1995).

In eukaryotes, the process is more complex and multiple factors are required for RNA polymerase to bind to the promoter. Under the influence of the nucleic acid sequence, the promoter may confer low, medium or high expression levels and may be constitutive or inducible.

Many constitutive promoters of bacillus have been described. Promoter Pveg of veg gene is a well described constitutively strong promoter. In addition, libraries of expression modules have been constructed which contain constitutive promoters from Bacillus with different promoter strengths (Guizou, S., et al (2016.) Nucleic Acids Res.44 (15), 7495-7508).

By adding an inducer molecule to the cell, the inducible promoter is activated or derepressed. Thus, the activator protein binds to the sequence immediately adjacent to the promoter sequence and actively recruits RNA polymerase and associated sigma factors to allow transcription to start. Well known examples are described of the alteration of the promoter conformation upon addition of arabinose and binding as a dimer to the operator site I ₁ And I ₂ Of (a) an araC-regulated Escherichia coli P _BAD The promoter and the bacillus mannose inducible promoter system PmanP regulated by an activator manR. Inducible promoters such as the lacUV5 promoter, the T7-phage promoter for expression in E.coli, and the Bacillus Pspac-I and Ppac-I promoters are negatively regulated by a lac repressor (encoded by the lacI gene) that binds to its specific lac operator site within (e.g., -35sigA recognition site and-10 sigA recognition site) or nearby (i.e., 3 'or 5' to the promoter sequence) the promoter sequence in the absence of an inducer molecule to prevent transcription. Another example is the PxylA inducible promoter system from Bacillus megaterium (Bacillus megaterium) which is widely used in Bacillus expression systems. The PxylA promoter is negatively regulated by the xylR repressor which comprises a xylR operator site 3' to the transcription initiation site.

Inducible promoter systems are generally advantageous for the cloning process in expression vectors, since the expression of genes under the control of such promoters is greatly reduced and thus the adverse effects, e.g. with regard to deprivation of cellular resources, interference with cellular metabolism, etc., are minimized, however, the adaptation to the desired protein expression needs to be carefully analyzed with regard to the number of inducer molecules added and the time point of inducible expression for each strain in which the promoter is used. In contrast, constitutive promoters have the advantage of being independent of the application of the inducer, do not require specific regulators or transporters, and are therefore active in a wide variety of bacteria.

Plasmids are extrachromosomal circular DNA that replicate autonomously in the host cell, and thus are not involved in host chromosomal replication.

For autonomous replication, the plasmid contains an origin of replication which makes autonomous replication of the vector in the host cell in question possible. Examples of bacterial origins of replication are the origins of replication of plasmids pUB110, pE194, pC194, pTB19, pAM β 1, pTA1060 which allow replication in Bacillus and the origins of replication of plasmids pBR322, colE1, pUC19, pSC101, pACYC177 and pACYC184 which allow replication in E.coli (Sambrook, J. And Russell, D.W. "molecular cloning Laboratory Manual", 3 rd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY.2001).

Plasmid copy number is defined as the average number of plasmids per bacterial cell or per chromosome under normal growth conditions. In addition, there are different types of origins of replication (also referred to as replicons) that result in different copy numbers in bacterial hosts.

The plasmid replicons pBS72 and pTB19 and the derivatives pTB51, pTB52 confer a low copy number, 6 copies and 1 to 8 copies, respectively, inside the bacillus cells, while the plasmids pE194 and pUB110 confer a low-medium copy number, 14-20 copies, respectively, per cell, and a medium copy number, 30-50 copies, respectively, per cell. The plasmid pE194 (Villafane, et al (1987): J. Bacteriol.169 (10), 4822-4829) was analyzed in more detail and several pE194-cop mutants with high copy numbers ranging from 85 to 202 copies inside the Bacillus were described. In addition, plasmid pE194 is temperature sensitive, up to 37 ℃ copy number stability, however in 43 ℃ above replication elimination. In addition, it presents a pE194 variant, called pE194ts, with 2 point mutations within the replicon region, resulting in a more exaggerated temperature sensitivity-copy number stabilization up to 32 ℃, whereas only 1 to 2 copies per cell at 37 ℃.

In E.coli, the pBR322 plasmid carrying the pMB1 replicon or its close relative, the colicin E1 (colE 1) replicon, maintains a low-to-moderate copy number, i.e.15-20 copies per bacterial cell. Deletion of the rop/rom gene inside the colE 1and pMB1 plasmid derivatives slightly increased the plasmid copy number inside the E.coli cells to a moderate copy number of 25-50. The pUC vector series is a small high copy plasmid with up to 200 copies per E.coli cell derived from a mutant pBR322 plasmid lacking the rop protein. The pUC plasmid is a well-established cloning vector due to its small size and high yield in plasmid preparation, compared to the pBR 322-derived and ColE 1-derived vectors mentioned above.

Alternatively, the p15A replicon present in the pACYC177/184 plasmid confers a low-to-medium copy number of 20 copies per cell and the pSC101 replicon confers a low copy number of 5-10 copies per cell. Plasmids with low to moderate copy numbers and encoding toxic or unfavorable expression constructs are usually stably maintained inside the cell, however, the yield is low when the plasmid is prepared. For subsequent transformation of bacterial cells, the amount of plasmid DNA becomes limiting compared to plasmid preparation of high copy plasmids. This is especially interesting for medium to high throughput applications when multiple preparations are performed in parallel.

The combination of plasmid copy number and promoter selection for gene expression determines the overall level of protein expression and therefore affects cell viability and plasmid stability.

CRISPR-based expression systems for gram-positive organisms (e.g. bacillus species) based on a single plasmid system approach, i.e. comprising a Cas9 endonuclease, a gRNA (e.g. sgRNA or crRNA/tracrRNA), repair homology sequences (donor DNA) on one single e.coli-bacillus shuttle vector, have been successfully applied.

Altenbuchner created a series of high copy pUC replicons that edited an e.coli-bacillus shuttle plasmid for bacillus subtilis (b.subtilis) based on the CRISPR/Cas9 genome, in combination with inducible promoters PmanP, pxylA, and PtetLM to express Cas9 endonuclease (Altenbuchner, (2016): applied and environmental microbiology 82 (17), 5421-5427). This allows highly efficient plasmid DNA preparation and stable maintenance within the E.coli cloning host. Likewise, a similar approach to construct a high copy pUC-derived CRISPRi-escherichia coli-Bacillus shuttle plasmid for use in Bacillus methanolicus (Bacillus methanolicus) was generated. The promoter of the B.methanolicus mannitol-activated gene mtlR driving the expression of defective Cas9 was modified by introducing a lacO site 3' to the promoter, thus effectively blocking transcriptional activity with intact lacI in E.coli ((ii)

Et al (2019), applied microbiology and biotechnology 103 (14), 5879-5889.

Another single plasmid approach to CRISPR/Cas9 application in bacillus subtilis uses a low to medium copy number replicon p15A to combine with the use of an inducer-independent promoter expressing Cas9 (a bacillus amyloliquefaciens (b. Amyloliquefaciens) PamyQ-amylase promoter), allowing successful cloning and stable maintenance of a CRISPR/Cas 9-based genome editing escherichia coli-bacillus shuttle plasmid in escherichia coli. A similar combination of a medium copy pBR 322-derived E.coli-Bacillus shuttle vector with Cas9 under the control of a constitutively strong promoter was used (Zhou et al (2019): international journal of biological macromolecules 122, 329-337).

Although low-copy and medium-copy backbones reduce the metabolic burden, this is accompanied by a decrease in the yield of plasmid from E.coli and prevents plasmid DNA from being isolated on the scale required for many transformation protocols that are difficult to transform Bacillus strains or to apply in high-throughput applications. Inducer-dependent promoter systems are not always suitable for use in a wide variety of different microorganisms and furthermore require analysis of the number of inducer-molecules and the point in time at which the promoter is induced. In addition, an additional promoter activation step by means of addition of an inducer molecule to the cell is required compared to constitutive promoters, which lengthens the overall time frame of the genome editing process.

There is therefore a need in the art to provide systems that allow the use of high copy vectors in combination with the use of constitutive promoters to overcome these limitations. One element of such systems is to provide constitutive promoters that confer reduced expression in bacteria that does not interfere, or does not significantly interfere, with the growth and/or vigor of the bacteria.

Detailed Description

A first embodiment of the invention comprises a method for producing one or more synthetic regulatory nucleic acid molecules conferring reduced constitutive expression as compared to a corresponding initial regulatory nucleic acid molecule in a bacterial cell, said method comprising the steps of

a. Identifying at least one initial regulatory nucleic acid molecule conferring constitutive expression in a bacterial cell, and

b. operably linking the initial regulatory nucleic acid molecule to a coding region encoding a protein that is heterologous with respect to the initial regulatory nucleic acid molecule, and

c. introducing into a vector a construct comprising said initiation regulatory nucleic acid molecule operably linked to a coding region, said vector comprising an origin of replication conferring a high copy number to said vector inside a bacterial cell, wherein said construct confers high expression of said coding region, wherein high expression of said coding region in a bacterial cell stresses said bacterial cell resulting in reduced or eliminated growth, and

d. transforming said vector into a bacterial cell, and

e. cultivating the transformed bacterial cell to recover a single clone, and

f. isolating a single clone that shows a growth rate comparable to that of a corresponding WT strain that can be isolated without said construct, and

g. isolating the construct from the clone; and is

h. Testing the synthetic regulatory nucleic acid molecule comprised in said construct for functional expression of a coding region operably linked to said synthetic regulatory nucleic acid molecule and optionally

i. Comparing the expression conferred by the synthetic regulatory nucleic acid with the expression conferred by the initial regulatory nucleic acid and optionally

j. Sequencing the corresponding regulatory nucleic acid molecules comprised in said construct, thereby identifying synthetic regulatory nucleic acid molecules conferring reduced constitutive expression in bacterial cells.

Reduced growth means that after incubation on a plate for a certain period of time under conditions suitable for the respective bacteria, a visible size difference of the respective colonies is visible between bacterial colonies comprising the construct as described above and bacterial colonies not comprising said construct. A bacterial colony comprising the construct will show smaller colonies as compared to a bacterial colony not comprising the construct. For example, E.coli bacteria will be incubated at 36-37 ℃ for 8-16 hours, after which the differences in colony size will be compared.

The coding region that stresses the bacteria expressing the coding region under the control of a constitutively strong promoter may for example be any coding region encoding a protein of greater than 150kDa, such as Cas9 or Cas12a, a coding region encoding an enzyme that induces DNA strand breaks or mutations, such as Cas9, cas12a and any other CRISPR Cas enzymes, homing endonucleases, meganucleases, adenosine deaminase or DNA glycosylases, a coding region encoding an enzyme that interferes with bacterial metabolism, such as an enzyme involved in the production of energy equivalents (ATP) or cofactors such as NADP, or a coding region encoding a transporter or transmembrane protein that interferes with substrate uptake or detoxification of bacterial cells.

Constitutive expression in bacterial cells means that the strength of expression derived from the corresponding promoter is substantially constant under a variety of conditions. In the present specification, constitutive expression means that expression derived from one promoter differs by less than 10-fold, preferably by less than 9-fold, preferably by less than 8-fold, preferably by less than 7-fold, preferably by less than 6-fold, preferably by less than 5-fold, preferably by less than 4-fold, more preferably by less than 3-fold, even more preferably by less than 2-fold under the following conditions: the exponential growth phase, transition phase and resting phase in rich medium, such as LB medium, rich medium substituted with a sugar, such as sucrose, lactose or glucose, preferably at a concentration of between 0.1% and 0.5%, preferably 0.3% glucose, and in minimal salt medium, such as M9 medium supplemented with a sugar, such as sucrose, lactose or glucose, preferably at a concentration of between 0.1% and 0.5%, preferably 0.3% glucose, under temperature conditions optimal for the respective cells.

To determine whether genes are differentially expressed, gene expression was measured across these conditions in at least triplicate and differences in these values were measured using the standard method of the art, DESeq2 software package (Love, m.i. et al, genome Biology 15 (12): 550 (2014)). This analysis will evaluate the observed fold change between conditions and the probability that this difference is due to random probability. Any gene that is more up-or/and down-regulated than defined above and has a probability of less than 5% due to random probability is considered differentially expressed and, therefore, not constitutively expressed.

Constitutive promoters are independent of other cellular regulatory factors and transcription initiation is dependent on sigma factor a (sigA). The sigA-dependent promoter comprises the sigma factor A specific recognition site '-35' -region and the '-10' -region.

Preferably, the constitutive promoter sequence is selected from the group consisting of: promoters Pveg, plepA, pserA, pymdA, pfba and derivatives thereof differing in the strength of gene expression (Guizou et al, (2016): nucleic Acids Res.44 (15), 7495-7508), phage SPO1 promoter P4, P5, P15 (WO 15118126), cryIIIA promoter from Bacillus thuringiensis (WO 3262 xzft 3262), combinations thereof, or active fragments or variants thereof.

An Origin of Replication (ORI) conferring a high copy number means an ORI which results in at least 51 copies of the corresponding vector in the corresponding bacterial cell in which the ORI is functional. Since the copy number depends on the temperature at which the corresponding bacteria are grown, it is preferred that this definition refers to the temperature at which the corresponding bacteria are grown in laboratories known to the skilled person as described for e.g.various strains (Bronikowski et al (2001): evolution 55 (1): 33-40).

Preferably for E.coli this means the copy number is measured at 36-37 ℃ growth, for Bacillus this means the copy number is measured at 36-37 ℃ growth.

One ORI conferring a medium copy number means maintaining 25-50 copies of the ORI vector, one conferring a low-medium copy number means maintaining 11-24 copies of the ORI per cell and one conferring a low copy number means maintaining 1-10 copies of the ORI vector inside the bacterial cell.

In a preferred embodiment, the e.coli ORI is selected from a high copy number ORI and the bacillus ORI is selected from a low copy number ORI, a low-medium copy number ORI and a medium copy number ORI.

More preferably, the E.coli ORI is selected from a high copy number ORI and the Bacillus ORI is selected from a low-to-medium copy number ORI.

More preferably, the E.coli ORI is selected from high copy number ORIs and the Bacillus ORI is selected from temperature sensitive low-medium copy number ORIs, e.g. derivatives of plasmid pE194 conferring low-medium copy number at 36-37 ℃ and low-medium copy number at 30-33 ℃ and non-replicating above 43 ℃.

More preferably, the E.coli ORI is selected from high copy number ORIs, such as, for example, pUC ORI, and the Bacillus ORI is selected from temperature sensitive low-medium copy number ORIs, such as, for example, derivatives of plasmid pE194ts conferring a low copy number at 36-37 ℃ and a low-medium copy number at 30-33 ℃ and non-replicating above 38 ℃.

The term "clone showing a comparable growth rate as a corresponding WT strain not comprising the construct" means a clone transformed with a construct as defined above, wherein said clone shows a growth rate of at least 50% of that of a WT bacterium when compared to a bacterium not comprising or transformed with such a construct. Preferably they have a growth rate of at least 60%, 65%, 70%, 75%, 80%, 85% as WT bacteria. More preferably, there is at least 90%, 95% growth rate as for WT bacteria such as or having the same growth rate as WT bacteria. The growth rate can be determined, for example, in terms of the cell density after a certain incubation time in liquid culture or in terms of the colony size on the plate.

Functional expression of a coding region means that expression of such a coding region is detectable at least, for example, by RNA detection methods such as RT-PCR, qPCR or by using detectable proteins such as fluorescent protein, GUS, an enzyme reaction specific for the respective enzyme or the gene deletion efficiency of the coding region encoding an enzyme that induces double strand breaks in the genome (e.g., CRISPR/Cas enzyme).

Yet another embodiment of the present invention is a method as defined above, wherein the synthetic regulatory nucleic acid molecule confers low to medium high expression in a bacterium distinct from the cell in which the recombinant nucleic acid is produced. For example, the initial regulatory nucleic acid molecule is tested and mutated in e.coli and later used for low to medium constitutive expression in Bacillus species (Bacillus sp.). For this purpose, the construct used in the method defined above may be cloned into a shuttle vector comprising a high copy number ORI for e.coli and another ORI for selection of bacillus species.

In a further embodiment of the invention, the synthetic regulatory nucleic acid molecule is in a cell of a gram-positive or gram-negative bacterium, preferably of the class Bacillus (Bacillus) or gamma-Proteobacteria (Gamma); more preferably in cells of the Bacillaceae family (Bacillaceae) or Enterobacteriaceae family (Enterobacteriaceae); even more preferably in the genus Bacillus (Bacillus) or Escherichia (Escherichia); and even more preferably in cells of the genus bacillus.

Preferred Bacillus cells include Bacillus alkalophilus (Bacillus alkalophilus), bacillus amyloliquefaciens, bacillus brevis (Bacillus brevis), bacillus circulans (Bacillus circulans), bacillus clausii (Bacillus clausii), bacillus coagulans (Bacillus coagulosus), bacillus firmus (Bacillus firmus), bacillus lautus (Bacillus lautus), bacillus lentus (Bacillus lentus), bacillus licheniformis, bacillus megaterium (Bacillus megaterium), bacillus pumilus (Bacillus pumilus), bacillus stearothermophilus (Bacillus stearothermophilus), bacillus methylotrophicus (Bacillus methylotrophicus), bacillus cereus (Bacillus cereus), bacillus parapsilosis (Bacillus amyloliquefaciens), bacillus subtilis, and Bacillus thuringiensis (Bacillus thuringiensis) cells.

Preferably, the synthetic regulatory nucleic acid molecule is active in cells of at least three different Bacillus species, in cells of at least two different Bacillus species, or in cells of at least one Bacillus species.

More preferably, the bacillus species comprises at least one of: bacillus subtilis, bacillus licheniformis (Bacillus licheniformis) or Bacillus pumilus (Bacillus pumilus). Most preferably, the synthetic regulatory nucleic acid molecule is active in a Bacillus licheniformis cell.

In yet another embodiment of the invention, any constitutively regulatory nucleic acid molecule active in bacteria conferring high expression may be used. Guiziou et al (Guiziou et al, (2016): nucleic Acids Res.44 (15), 7495-7508) describe various regulatory Nucleic acid molecules suitable for the methods of the invention and further describe how to identify additional regulatory Nucleic acid molecules suitable for the methods of the invention. Preferably, the initial regulatory nucleic acid molecule conferring constitutive expression in a bacterial cell is selected from

a) 28 and 29 of the amino acid sequences of SEQ ID NO,

b) A nucleic acid molecule comprising at least 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs identical to 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs of the sequence depicted in SEQ ID NO 28 or 29, and

c) A nucleic acid molecule having at least 90% identity, preferably at least 91%, 92%, 93%, 94% or 95%, more preferably at least 96%, 97%, 98% or 99% identity over the entire length of the sequence depicted in SEQ ID NO 28 or 29, and

d) Nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of at least 20 consecutive base pairs, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs of a nucleic acid molecule according to SEQ ID NO 28 or 29,

e) The complement of any of the nucleic acid molecules as defined in a) to d).

Yet another embodiment of the invention is a synthetic regulatory nucleic acid molecule, wherein the regulatory nucleic acid molecule is comprised in the group consisting of

A) A nucleic acid molecule having the sequence of

SEQ ID NO

35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

b) 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs, and at least 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs identical to 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs, and

c) A nucleic acid molecule having at least 90% identity, preferably at least 91%, 92%, 93%, 94% or 95%, more preferably at least 96%, 97%, 98% or 99% identity, over the entire length, to a sequence according to

SEQ ID NO

35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

d) A nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of at least 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs of the nucleic acid molecule of any one of

SEQ ID NO

35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47 and

e) The complement of any of the nucleic acid molecules as defined in A) to D),

wherein the sequence as defined in B) to E) differs from the corresponding initial regulatory nucleic acid molecule having SEQ ID NO 28 or 29 and preferably comprises at least one base deletion or insertion compared to the corresponding initial regulatory nucleic acid.

Yet another embodiment of the invention is a synthetic regulatory nucleic acid molecule as described above, wherein the nucleic acid molecule is produced using a method as defined above.

Expression constructs comprising synthetic regulatory nucleic acid molecules as defined above are also an embodiment of the invention. Preferably, the expression construct comprises a synthetic regulatory nucleic acid molecule and is functionally linked to a coding region encoding a CRISPR/Cas protein, meganuclease protein or TALE/N, preferably a CRISPR/Cas protein that is a Cas9 or Cas12a protein.

A vector comprising a synthetic regulatory nucleic acid molecule as defined above or an expression construct as defined above is a further embodiment of the invention.

Yet another embodiment of the invention is a microorganism comprising a regulatory nucleic acid molecule or expression construct or vector as defined above.

Definition of

Abbreviations: GFP-green fluorescent protein, GUS- β -galactosidase, BAP-6-benzylaminopurine, 2,4-D-2,4-dichlorophenoxyacetic acid, MS-Murashige-Skoog medium, NAA-1-naphthylacetic acid, MES,2- (N-morpholino) -ethanesulfonic acid, IAA: indoleacetic acid, kan: kanamycin sulfate, GA 3-gibberellic acid, timentin ^TM : ticarcillin disodium/clavulanate potassium, micro: microliter.

It is to be understood that this invention is not limited to the particular methodology or protocol. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, and so forth. The term "about" is used herein to mean about, approximately, left-right, and within the range of … …. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the limits above and below the stated values. Generally, the term "about" is used herein to modify a numerical value above and below the stated value by a variation of 20%, preferably above or below (higher or lower) 10%. As used herein, the word "or" means any one member of a particular list and also includes any combination of the listed members. The words "comprise," "comprising," "include," "including," and "includes" when used in this specification and in the following claims are intended to specify the presence of one or more stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof. For clarity, certain terms used in this specification are defined and used as follows.

Coding region: as used herein, the term "coding region" when used in reference to a structural gene refers to a nucleotide sequence that encodes the amino acids found in a nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded on the 5 'side by the nucleotide triplet "ATG" coding for the initiator methionine and on the 3' side by the three triplets specifying the stop codon (i.e., TAA, TAG, TGA). Alternatively, a nucleotide triplet may be "GTG" or "TTG" and is considered as an initial nucleotide triplet in that the ribosome binding site (Shine Dalgarno) is present from 4 nucleotides to 12 nucleotides apart relative to 5' of the nucleotide triplet. The genomic form of the gene may also comprise sequences located at the 5 '-and 3' -ends of the sequences present in the RNA transcript. These sequences are referred to as "flanking" sequences or regions (which flanking sequences are located 5 'or 3' to the untranslated sequences present on the mRNA transcript). The 5' -flanking region may contain regulatory sequences such as promoters and enhancers which control or influence gene transcription, and a ribosome binding site (Shine Dalgarno) which controls or influences translation of mRNA. The 3' -flanking region may contain sequences that direct transcription termination and post-transcriptional cleavage.

Complementary: "complementary" or "complementarity" refers to two nucleotide sequences comprising antiparallel nucleotide sequences that are capable of pairing with each other (by the base pairing rules) upon formation of hydrogen bonds between complementary base residues in the antiparallel nucleotide sequences. For example, the sequence 5'-AGT-3' is complementary to the sequence 5 '-ACT-3'. Complementarity may be "partial" or "total". "partial" complementarity is where one or more nucleic acid bases are unmatched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acid molecules is where each nucleic acid base matches another base according to the base pairing rules. The degree of complementarity between nucleic acid molecule chains has a significant effect on the efficiency and strength of hybridization between nucleic acid molecule chains. A nucleic acid sequence "complement" as used herein refers to a nucleotide sequence whose nucleic acid molecules exhibit full complementarity to the nucleic acid molecules of the nucleic acid sequence.

Donor DNA molecule: as used herein, the terms "donor DNA molecule", "repair DNA molecule" or "template DNA molecule" are all used interchangeably herein to mean a DNA molecule having a sequence to be introduced into the genome of a cell. It may be flanked at the 5 'and/or 3' end by sequences homologous or identical to the sequences in the target region of the genome of said cell. It may comprise sequences not naturally present in the corresponding cell such as ORFs, non-coding RNAs or regulatory elements which should be introduced to the target region or it may comprise sequences homologous to the target region except for at least one mutation (gene editing): the sequence of the donor DNA molecule may be added to the genome or it may replace the sequence of the length of the donor DNA sequence in the genome.

Double-stranded RNA: a "double-stranded RNA molecule" or "dsRNA" molecule comprises a sense RNA fragment of a nucleotide sequence and an antisense RNA fragment of the nucleotide sequence, both comprising nucleotide sequences that are complementary to each other, thereby allowing the sense RNA fragment and the antisense RNA fragment to pair and form a double-stranded RNA molecule.

Endogenous: an "endogenous" nucleotide sequence refers to a nucleotide sequence that is present in the genome of an untransformed cell.

Expressing: "expression" refers to the biosynthesis of a gene product, preferably to the transcription and/or translation of a nucleotide sequence, such as an endogenous gene or a heterologous gene, in a cell. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and optionally subsequent translation of the mRNA into one or more polypeptides. In other cases, expression may refer only to transcription of DNA carrying an RNA molecule.

Expression construct: an "expression construct" as used herein means a DNA sequence capable of directing the expression of a specific nucleotide sequence in a suitable plant part or plant cell, which DNA sequence comprises a promoter functional in said plant part or plant cell into which this DNA sequence is to be introduced, said promoter being operably linked to a nucleotide sequence of interest optionally operably linked to a termination signal. If translation is required, the DNA sequence will generally also comprise sequences required for correct translation of the nucleotide sequence. The coding region may encode a protein of interest, but may also encode a functional RNA of interest, e.g. RNAa, siRNA, snoRNA, snRNA, microRNA, ta-siRNA or any other non-coding regulatory RNA, in sense or antisense orientation. An expression construct comprising a nucleotide sequence of interest may be chimeric, meaning that one or more of the components of the expression construct are heterologous with respect to one or more of the other components of the expression construct. The expression construct may also be one which occurs naturally but which has been obtained in recombinant form for heterologous expression. However, in general, the expression construct is heterologous with respect to the host, i.e., the particular DNA sequence of the expression construct does not naturally occur in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. Expression of the nucleotide sequence in the expression construct cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. In terms of cellular development, the promoter may also be specific for a particular developmental stage (e.g., biofilm formation, sporulation).

External application: the term "foreign" refers to any nucleic acid molecule (e.g., a gene sequence) that is introduced into the genome of a cell by experimental manipulation and can comprise a sequence present in the cell, so long as the introduced sequence contains some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) and thus differs relative to the naturally occurring sequence.

Functional connection: the term "functionally linked" or "functionally linked" is understood to mean, for example, that a regulatory element (e.g. a promoter) is associated with a nucleic acid sequence to be expressed and, if desired, is arranged in succession with other regulatory elements in such a way that each regulatory element can fulfill its function of purpose in order to allow, modify, facilitate or influence the expression of said nucleic acid sequence. As a synonym, "operatively linked" or "operatively linked" may be used. Depending on the arrangement of the nucleic acid sequences, expression may result in sense or antisense RNA. For this purpose, direct linkage in the chemical sense is not necessarily required. Genetic control sequences, such as enhancer sequences, can also exert their effect on the target sequence from remote locations or even from other DNA molecules. A preferred arrangement is one in which the nucleic acid sequence to be expressed is recombinantly located behind the sequence acting as promoter, so that the two sequences are covalently linked to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs. In a preferred embodiment, the nucleic acid sequence to be transcribed is located after the promoter in such a way that the transcription start is identical to the desired start of the chimeric RNA of the invention. Functional recombinant constructs can be generated by means of conventional recombination techniques such as described (e.g., in Maniatis T, fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory, cold Spring Harbor (NY); silhavy et al (1984) Experiments with Gene Fusions, cold Spring Harbor Laboratory, cold Spring Harbor (NY); ausubel et al (1987) Current Protocols in Molecular Biology, grne Publishing. And. Wilinterference; gelvin et al (eds.) (1990) Plant Molecular Biology Manual; kluwer Acedification publication, network, the Dordey, the The). However, other sequences, for example a sequence which acts as a linker with a specific cleavage site for a restriction enzyme or as a signal peptide, may also be located between these two sequences. Insertion of the sequence may also result in expression of the fusion protein. Preferably, the expression construct consisting of the linkage of the regulatory region, e.g.the promoter, and the nucleic acid sequence to be expressed may be present in vector-integrated form and inserted into the plant genome, for example by transformation.

Gene: the term "gene" refers to a region operably linked to a suitable regulatory sequence capable of regulating the expression of a gene product (e.g., a polypeptide or functional RNA) in some manner. Genes include untranslated regulatory regions (e.g., promoters, enhancers, repressors, etc.) in the DNA before (upstream) and after (downstream) the coding regions (open reading frames, ORFs), as well as intervening sequences (e.g., introns) between individual coding regions (e.g., exons) as desired. The term "structural gene" as used herein is intended to refer to a DNA sequence that is transcribed into mRNA which is subsequently translated into an amino acid sequence characteristic of a particular polypeptide.

"Gene editing" as used herein means the introduction of a particular mutation at a particular location in the genome of a cell. Gene editing can be introduced by a precise editing process using more advanced techniques such as using CRISPR Cas systems and donor DNA or CRISPR Cas systems associated with mutagenic activities such as deaminases (WO 15133554, WO 17070632).

Genome and genomic DNA: the term "genome" or "genomic DNA" refers to the heritable information of a host organism. In eukaryotes, the genomic DNA includes DNA of the nucleus (also referred to as chromosomal DNA), and also includes DNA of the plastids (e.g., chloroplasts) and other organelles (e.g., mitochondria). Preferably, the term "genome" or genomic "DNA" refers to chromosomal DNA of the nucleus. In prokaryotes, the genomic DNA includes chromosomal DNA inside bacterial cells.

Heterologous: the term "heterologous" with respect to a nucleic acid molecule or DNA refers to a nucleic acid molecule that is operably linked to or manipulated to become operably linked to a second nucleic acid molecule, e.g., a promoter, that is not operably linked to the nucleic acid molecule in nature (e.g., in the genome of a wild-type (WT) plant) or that is operably linked to the nucleic acid molecule at a different location or position in nature (e.g., in the genome of a WT plant).

Preferably, the term "heterologous" in the context of a nucleic acid molecule or DNA (e.g., NEENA) refers to a nucleic acid molecule that is operably linked or manipulated to become operably linked to a second nucleic acid molecule that is not operably linked to in nature, e.g., a promoter.

Heterologous expression constructs comprising a nucleic acid molecule and one or more regulatory nucleic acid molecules linked thereto, such as a promoter or a transcription termination signal, are, for example, constructs resulting from experimental manipulations in which a) the nucleic acid molecule or b) the regulatory nucleic acid molecule or c) both, i.e. (a) and (b), are not located in their natural (native) genetic environment or have been modified by experimental manipulations, examples of modifications being substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues. A natural genetic environment refers to a natural chromosomal locus in the source organism or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the sequence of the nucleic acid molecule is preferably retained, at least partially retained. The environment is distributed on at least one side of the nucleic acid sequence and has a sequence length of at least 50bp, preferably at least 500bp, particularly preferably at least 1,000bp, very particularly preferably at least 5,000bp. Naturally occurring expression constructs, e.g., naturally occurring combinations of promoters and corresponding genes-become transgenic expression constructs when modified by non-natural synthetic "artificial" methods, e.g., mutagenesis. Such methods have been described (U.S. Pat. No. 5,565,350, WO 00/15815. For example, a nucleic acid molecule encoding a protein operably linked to a promoter that is not the native promoter of the nucleic acid molecule is considered heterologous with respect to the promoter. Preferably, the heterologous DNA is not endogenous or not naturally associated with the cell relative to the cell into which it is introduced, but has been obtained from another cell or has been synthesized. Heterologous DNA also includes DNA sequences that contain some modification of the endogenous DNA sequence, multiple copies of the endogenous DNA sequence that do not naturally occur or are naturally not joined to another DNA sequence that is physically linked to the DNA sequence. Typically, although not necessarily, the heterologous DNA encodes an RNA or protein not normally encoded by the cell into which it is introduced.

The term "hybridization" as defined herein is a process in which substantially complementary nucleotide sequences renature with each other. The hybridization process can be carried out completely in solution, i.e.both complementary nucleic acids are in solution. The hybridization process can also take place with one of the complementary nucleic acids immobilized to a medium such as magnetic beads, sepharose beads or any other resin. The hybridization process can also be carried out with one of the complementary nucleic acids immobilized on a solid support such as a nitrocellulose or nylon membrane or on a support, including but not limited to a silicate glass support (the latter being referred to as a nucleic acid array or microarray or as a nucleic acid chip), by, for example, photolithography. To allow hybridization to occur, the nucleic acid molecules are typically thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single-stranded nucleic acids.

This formation or melting of the hybrid molecule depends on a variety of parameters, including but not limited to temperature. An increase in temperature favors melting, while a decrease in temperature favors hybridization. However, this hybrid molecule formation process does not vary in a linear manner with applied temperature: the hybridization process is dynamic and the already formed nucleotide pairs also support adjacent nucleotide pairing. Thus, in good approximation, hybridization is a yes or no process, and there exists a temperature that essentially defines the boundary between hybridization and no hybridization. This temperature is the melting temperature (Tm). The Tm is the temperature in degrees celsius at which 50% of all molecules with a given nucleotide sequence hybridize to double strands and 50% exist as single strands.

The melting temperature (Tm) depends on the physical properties of the nucleic acid sequence being analyzed and thus can indicate the relationship between two different sequences. However, the melting temperature (Tm) is also influenced by various other parameters that are completely unrelated to the sequence, and the applied hybridization experimental conditions must be considered. For example, increasing the salt (e.g., monovalent cation) results in a higher Tm.

The Tm for a given hybridization condition can be determined by performing physical hybridization experiments, but the Tm for a given pair of DNA sequences can also be estimated in silico. In this embodiment, the equation of Meinkoth and Wahl (anal. Biochem.,138, 267-284,1984) is used for fragments having a length of 50 or more bases: tm =81.5 ℃ +16.6 (log M) +0.41 (% GC) -0.61 (formamide%) -500/L.

M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine in the DNA fragment,% formamide is the percentage formamide in the hybridization solution, and L is the length of the hybrid molecule in base pairs. This equation applies to the salt range of 0.01 to 0.4M and GC% of 30% to 75%.

Although the above Tm is the temperature of a perfectly matched probe, the Tm is reduced by about 1 ℃ for every 1% mismatch (Bonner et al, J.mol.biol.81:123-135, 1973): tm = [81.5 ℃ +16.6 (log M) +0.41 (% GC) -0.61 (% formamide) -500/L ] -non-identity%.

This equation can be used for probes having 35 or more nucleotides and is widely used in the scientific methods literature (e.g., cited in "Recombinant DNA Principles and methods", james Greene, section "Biochemistry of Nucleic acids", paul S.Miller, page 55; 1998, CRC Press), numerous patent applications (e.g., cited in: US 7026149) and also in data files from commercial enterprises (e.g., "acquistions for practical Tm" from www.genomics.agilent.com).

Other formulas for calculating Tm that are less preferred in this embodiment are only possible for the case shown:

for DNA-RNA hybrid molecules (Casey, j. And Davidson, n. (1977) Nucleic Acids res.,4:

tm =79.8 ℃ +18.5 (log M) +0.58 (% GC) +11.8 (% GC x% GC) -0.5 (formamide%) -820/L.

For RNA-RNA hybrid molecules (Bodkin, d.k. and Knudson, d.l. (1985) j.virol.methods,10:

tm =79.8 ℃ +18.5 (log M) +0.58 (% GC) +11.8 (% GC x% GC) -0.35 (formamide%) -820/L.

For oligonucleotide probes of less than 20 bases (Wallace, R.B. et al (1979) Nucleic Acid Res.6: 3535): tm =2x n (a + T) +4x n (G + C), where n is the number of corresponding bases in the probe forming the hybrid molecule.

For 20-35 nucleotide oligonucleotide probes, a modified Wallace calculation may be applied: tm =22+1.46n (A + T) +2.92n (G + C), where n is the number of corresponding bases in the probe forming the hybrid molecule.

For other oligonucleotides, a nearest neighbor model for calculating melting temperatures should be used, along with appropriate thermodynamic data:

Tm＝(∑(ΔHd)+ΔHi)/(∑(ΔSd)+ΔSi+ΔSself+R×ln(cT/b))+16.6log[Na+]–273.15(Breslauer,K.J.,Frank,R.,

h. Marky, l.a.1986predicting DNA duplex stability from the base sequence (prediction of DNA duplex stability from base sequence). Proc.natl acad.sci.usa 833746-3750; alejandro Panjkovich, francisco Mel, 2005.Comparison of differential melting calculation methods for short DNA sequences Bioinformatics,21 (6): 711-722)

Wherein:

tm is the melting temperature in degrees Celsius;

Σ (Δ Hd) and Σ (Δ Sd) (accordingly) are the enthalpy sum and entropy sum calculated for all internal nearest neighbor doublets;

Δ Sself is the entropy penalty for self-complementary sequences;

Δ Hi and Δ Si are the sum of the starting enthalpy and starting entropy, respectively;

r is a gas constant (fixed at 1,987 cal/K.mol);

cT is the total chain concentration in molar units;

the constant b assumes a value of 4 for non-self-complementary sequences, or a duplex for self-complementary strands or a duplex for which there is a significant excess of one of the strands, which equals 1.

Thermodynamic calculation assumptions: renaturation occurs in a buffered solution near a pH of 7.0 and a two-state transition occurs.

The thermodynamic values used for this calculation can be obtained from Table 1 of (Alejandro Panjkovich, francisco Mel, 2005. Complex of differential measuring calculation methods for short DNA sequences. Bioinformatics,21 (6): 711-722), or from the original research theoryText (Breslauer, k.j., frank, r.,

h. Marky, l.a.1986predicting DNA duplex stability from the base sequence (prediction of DNA duplex stability from base sequence). Proc.natl acad.sci.usa 833746-3750; santalocia, j., jr, allwi, h.t., seneviratne, p.a.1996improved near-neighbor parameters for predicting DNA duplex stability, biochemistry 353555-3562; sugimoto, n., nakano, s., yoneyama, m., honda, k.1996improved thermodynamic parameters and helix initiation factors for predicting DNA duplex stability.

To estimate Tm according to this embodiment in silico, a set of bioinformatic sequence alignments is first generated between two sequences. Such alignments can be generated by various tools known to those skilled in the art, such as the programs "Blast" (NCBI), "Water" (EMBOSS) or "mather" (EMBOSS) that generate local alignments, or the program "Needle" (EMBOSS) that generates global alignments. These tools should be applied with their default parameter set, as well as with some parameter variation. For example, the program "MATCHER" can be applied with the various parameters of gap opening/gap extension (such as 14/4/14/5/20/10/30/5. For example, blastN (NCBI) can be applied in conjunction with increased e-value cutoffs (e.g., e +1 or even e + 10) with the aim of also identifying very short alignment results, particularly in small-format databases.

It is important to consider local alignments, since hybridization may not necessarily occur over the entire length of the two sequences, but may occur optimally in different regions, which then determine the actual melting temperature. Thus, from the total alignment generated, the alignment length, the GC content of the alignment (% GC content of matching bases within the alignment, in a more precise manner), and the alignment identity must be determined. Subsequently, the predicted melting temperature (Tm) must be calculated for each alignment. The highest calculated Tm is used to predict the actual melting temperature.

The term "hybridizes within the complete sequence of the invention" as defined herein means that for sequences of more than 300 bases in length, each fragment must hybridize when the sequences of the invention are fragmented into small pieces of about 300 to 500 bases in length. For example, the DNA may be fragmented by using one restriction enzyme or a combination of restriction enzymes. Bioinformatic computer simulation calculation of Tm was performed by the same procedure as described above, only for each fragment. The actual hybridization of the individual fragments can be analyzed by standard southern blot analysis or comparable methods known to those skilled in the art.

The term "stringency" as defined herein describes the ease with which a hybrid molecule may be formed between two nucleotide sequences. Conditions of "higher stringency" require more bases of one sequence to pair with another sequence (conditions of "higher stringency" in which the melting temperature, tm, is reduced), and conditions of "lower stringency" allow some more bases to unpair. Thus, the degree of relationship between two sequences can be estimated by the actual stringency conditions under which they are still capable of forming hybrid molecules. Increasing stringency can be achieved by keeping the experimental hybridization temperature constant and reducing the salt concentration, or by keeping the experimental hybridization temperature constant and increasing the experimental hybridization temperature, or a combination of these parameters. In addition, increasing formamide increases stringency. The skilled artisan is aware of additional parameters that can be altered during hybridization and that stringency conditions will be maintained or altered (Sambrook et al (2001) Molecular Cloning: a Laboratory manual, 3 rd edition, cold Spring Harbor Laboratory Press, CSH, new York or Current Protocols in Molecular Biology, john Wiley & Sons, N.Y. (1989 and annual updates)).

Common hybridization experiments were performed by an initial hybridization step, followed by one to several wash steps. The solutions used for these steps may contain additional components such as EDTA, SDS, fragmented sperm DNA or similar reagents that prevent degradation of the analytical sequences and/or prevent non-specific background binding of the probes as known to those skilled in the art (Sambrook et al (2001) Molecular Cloning: a Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press, CSH, new York or Current Protocols in Molecular Biology, john Wiley & Sons, N.Y. (1989 and annual updates)).

The usual probes for hybridization experiments are generated by a random priming labeling method, originally developed by Feinberg and Vogelstein (anal. Biochem.,132 (1), 6-13 (1983); anal. Biochem.,137 (1), 266-7 (1984) and based on the hybridization of a mixture of all possible hexanucleotides with the DNA to be labeled the labeled probe product will in fact be a set of fragments of variable length, typically in the size range of 100-1000 nucleotides with a maximum fragment concentration typically of about 200 to 400bp.

For the present invention, the sequences described herein are analyzed by hybridization experiments in which probes are generated from another sequence, and such probes are generated by standard random priming labeling methods. For the present invention, the probe consists of a set of labeled oligonucleotides having a size of about 200-400 nucleotides. Hybridization between a sequence of the invention and another sequence means that hybridization of the probe occurs within the complete sequence of the invention, as defined above. Hybridization experiments were performed by achieving the highest stringency according to the stringency of the final wash step. The final wash step has stringency conditions comparable to those of the following wash conditions: at least washing condition 1: 1.06x SSC, 0.1% SDS, 0% formamide at 50 ℃; in another embodiment at least wash conditions 2: 1.06x SSC, 0.1% SDS, 0% formamide at 55 ℃; in another embodiment at least wash condition 3: 1.06x SSC, 0.1% SDS, 0% formamide at 60 ℃; in another embodiment at least wash conditions 4: 1.06x SSC, 0.1% SDS, 0% formamide at 65 ℃; in another embodiment at least wash conditions 5: 0.52x SSC, 0.1% SDS, 0% formamide at 65 ℃; in another embodiment at least wash conditions 6: 0.25x SSC, 0.1% SDS, 0% formamide at 65 ℃; in another embodiment at least washing condition 7: 0.12x SSC, 0.1% SDS, 0% formamide at 65 ℃; in another embodiment at least wash condition 8: 0.07 XSSC, 0.1% SDS, 0% formamide at 65 ℃.

A "low stringency wash" has stringency conditions which are comparable to those of at least wash condition 1, but not more stringent than wash condition 3, wherein the wash conditions are as described above.

A "high stringency wash" has stringency conditions that are comparable to at least washing condition 4, in another embodiment at least washing condition 5, in another embodiment at least washing condition 6, in another embodiment at least washing condition 7, in another embodiment at least washing condition 8, wherein washing conditions are as described above.

"identity": when used in comparing two or more nucleic acid or amino acid molecules, "identity" means that the sequences of the molecules share some degree of sequence similarity, i.e., the sequences are partially identical.

Enzyme variants may be defined by their sequence identity when compared to the parent enzyme. Sequence identity is typically provided as a "percent sequence identity" or a "percent identity". To determine the percent identity between two amino acid sequences, in a first step, a paired sequence alignment is generated between the two sequences, wherein the two sequences are aligned over their entire length (i.e., paired global alignment). The alignment results were generated with a program implementing the Needlem and Wunsch algorithm (j.mol.biol. (1979) 48, pages 443-453), preferably by using the program "needlel" (European Molecular Biology Open Software Suite (EMBOSS)), with program default parameters (gap opening =10.0, gap extension =0.5 and matrix = EDNAFULL).

The following examples are intended to illustrate two nucleotide sequences, but the same calculations apply to protein sequences:

seq A: AAGATACTG length: 9 bases

Seq B: gattctga length: 7 bases

Thus, the shorter sequence is sequence B.

Generating pairwise global alignments showing both sequences over their entire length yields

The "I" symbol in the alignment indicates the same residue (which means the base of DNA or the amino acid of protein). The number of identical residues is 6.

The "-" symbol in the alignment indicates a vacancy. The number of vacancies introduced by the alignment within Seq B is 1. The number of vacancies introduced by alignment is 2 at the boundary of Seq B and 1 at the boundary of Seq a.

The aligned length of the aligned sequences is shown to be 10 over its entire length.

Pairwise alignments according to the invention that produce sequences that exhibit a shorter length over their entire length thus yield:

the pairwise alignment according to the invention which results in the display of sequence A over its entire length thus results:

pairwise alignments according to the invention are generated showing sequence B over its entire length thus generating:

the alignment length of the shorter sequence was shown to be 8 over its entire length (there was a gap to be considered in the alignment length of the shorter sequence).

Thus, it is shown that the alignment length of Seq A will be 9 over its entire length (meaning that Seq A is a sequence of the invention).

Thus, it is shown that the alignment length of Seq B will be 8 over its entire length (meaning that Seq B is a sequence of the invention).

After aligning the two sequences, in a second step, an identity value is determined from the resulting alignment. For the purposes of this specification, percent identity is calculated by: % = (identical residues/length of aligned region showing the corresponding sequence of the invention over its entire length) = 100. Thus, according to this embodiment, sequence identity is calculated in connection with comparing two amino acid sequences by dividing the number of identical residues by the length of the aligned region over which the corresponding sequences of the invention are displayed. This value is multiplied by 100 to yield the "% identity". According to the examples provided above,% identity: for Seq a (6/9) × 100=66.7% as inventive sequence; for Seq B, (6/8) × 100=75% as the sequence of the invention.

Indel (insertion/deletion) is a term for the random insertion or deletion of bases in the genome of an organism that are involved in the repair of DSB by NHEJ. It is classified among small genetic variations, measuring 1 to 10 000 base pairs in length. As used herein, it refers to the random insertion or deletion of a base in or immediately adjacent to a target site (e.g., less than 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, 50bp, 40bp, 30bp, 25bp, 20bp, 15bp, 10bp, or 5bp upstream and/or downstream thereof).

By introducing the donor DNA molecule in the target site of the target DNA, the terms "introducing", "introducing" and the like mean any introduction of the sequence of the donor DNA molecule into the target region or introduction of the sequence of the donor DNA molecule or part thereof into the target region where the donor DNA is used as a polymerase template, e.g. by physically integrating the donor DNA molecule or part thereof into the target region.

Syngeneic: organisms (e.g., plants) that are genetically identical, except that they may differ by the presence or absence of a heterologous DNA sequence.

Separating: the term "isolated" as used herein means that the material has been removed by hand and is present from its original natural environment and is therefore not a product of nature. An isolated material or molecule (such as a DNA molecule or enzyme) may exist in a purified form or may exist in a non-native environment such as, for example, in a transgenic host cell. For example, a naturally occurring polynucleotide or polypeptide present in a living plant is not isolated, whereas the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition, and isolated in that such vector or composition is not part of its original environment. Preferably, the term "isolated" when used in connection with a nucleic acid molecule, such as in an "isolated nucleic acid sequence," refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its natural source. An isolated nucleic acid molecule is one that exists in a form or environment that is different from the form or environment in which it is found in nature. In contrast, an unseparated nucleic acid molecule is a nucleic acid molecule such as DNA and RNA that is found in the state in which it exists in nature. For example, a given DNA sequence (e.g., gene) is found in the vicinity of adjacent genes on the host cell chromosome; RNA sequences, such as a particular mRNA sequence encoding a particular protein, are found in cells as a mixture with numerous other mrnas encoding various proteins. However, an isolated nucleic acid sequence comprising, for example, SEQ ID NO. 12, includes by way of example such nucleic acid sequences that normally contain SEQ ID NO. 12 in a cell, where the nucleic acid sequence is at or otherwise flanked by a different chromosomal location than that of a natural cell. An isolated nucleic acid sequence may exist in single-stranded or double-stranded form. When an isolated nucleic acid sequence is used to express a protein, the nucleic acid sequence will contain at least a portion of the sense or coding strand (i.e., the nucleic acid sequence may be single-stranded). Alternatively, it may contain both the sense and antisense strands (i.e., the nucleic acid sequence may be double stranded).

Non-coded: the term "non-coding" refers to a sequence in a nucleic acid molecule that does not encode part or all of the expressed protein. Non-coding sequences include, but are not limited to, introns, enhancers, promoter regions, 3 'untranslated regions, and 5' untranslated regions.

Nucleic acids and nucleotides: the terms "nucleic acid" and "nucleotide" refer to naturally occurring or synthetic or artificial nucleic acids or nucleotides. The terms "nucleic acid" and "nucleotide" include deoxyribonucleotides or ribonucleotides or any nucleotide analogue and polymer or hybrid thereof in either single-or double-stranded, sense or antisense form. Unless otherwise indicated, a particular nucleic acid sequence also inherently includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used herein interchangeably with "gene", "cDNA", "mRNA", "oligonucleotide", and "polynucleotide". Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar, and/or phosphate, including but not limited to 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, 5-bromo-uracil substitutions, and the like; and 2 '-position sugar modifications, including but not limited to sugar modified ribonucleotides, wherein the 2' -OH is replaced by a group selected from H, OR, halogen, SH, SR, NH2, NHR, NR2, or CN. Short hairpin RNAs (shrnas) may also contain unnatural elements such as unnatural bases, e.g., inosine and xanthine, unnatural sugars, e.g., 2' -methoxyribose, or unnatural phosphodiester bonds, e.g., methyl phosphate, phosphorothioate, and peptide.

Nucleic acid sequence: the phrase "nucleic acid sequence" refers to a single-or double-stranded polymer of deoxyribonucleotides or ribonucleotides read from the 5 '-end to the 3' -end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA, and DNA or RNA that exerts a mainly structural role. "nucleic acid sequence" also refers to a contiguous string of abbreviations, letters, characters or words that represent nucleotides. In one embodiment, a nucleic acid may be a "probe," which is a relatively short nucleic acid, typically less than 100 nucleotides in length. Often, nucleic acid probes have a length of about 50 nucleotides to about 10 nucleotides. A "target region" of a nucleic acid is the portion of the nucleic acid that is identified as having a target. A "coding region" of a nucleic acid is that portion of the nucleic acid which, when placed under the control of appropriate regulatory sequences, is transcribed and translated in a sequence-specific manner to produce a particular polypeptide or protein. The coding region is said to encode such a polypeptide or protein.

Oligonucleotide: the term "oligonucleotide" refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, as well as oligonucleotides having non-naturally occurring portions that function similarly. Such modified or substituted oligonucleotides are often preferred over their native forms for desirable properties such as enhanced cellular uptake, enhanced nucleic acid target affinity, and increased stability in the presence of nucleases. The oligonucleotide preferably comprises two or more nucleotide monomers (nucleomonomers) covalently coupled to each other by a bond (e.g., a phosphodiester bond) or a substituted bond (substitate linkage).

Overhang: an "overhang" is a relatively short single-stranded nucleotide sequence (also referred to as an "extension," "extended end," or "sticky end") on the 5 '-or 3' -hydroxyl end of a double-stranded oligonucleotide molecule.

Polypeptide: the terms "polypeptide", "peptide", "oligopeptide", "polypeptide", "gene product", "expression product" and "protein" are used interchangeably herein to refer to a polymer or oligomer of contiguous amino acid residues.

Preproprotein: a protein that is normally targeted to an organelle such as a chloroplast and still comprises its transit peptide.

By "exactly" with respect to the introduction of the donor DNA molecule in the target region is meant that the sequence of the donor DNA molecule is introduced into the target region without any insertions/deletions, duplications or other mutations, as compared to the unaltered DNA sequence of the target region that is not comprised in the sequence of the donor DNA molecule.

Primary transcript: the term "primary transcript" as used herein refers to an immature RNA transcript of a gene. "primary transcript" for example still contains introns and/or still contains no poly-a tail or cap structure and/or lacks other modifications, such as trimming or editing, necessary for its normal functioning as a transcript.

A "promoter" or "promoter sequence" or "regulatory nucleic acid" is a nucleotide sequence located upstream of a gene on the same strand as the gene that is capable of effecting transcription of the gene. The promoter is followed by the transcription start site of the gene. The promoter is recognized by RNA polymerase (along with any desired transcription factors), which initiates transcription. A functional fragment or functional variant of a promoter is a nucleotide sequence that is recognized by RNA polymerase and is capable of initiating transcription.

Purification of: as used herein, the term "purified" refers to a molecule, i.e., a nucleic acid sequence or an amino acid sequence, that is removed, isolated, or separated from its natural environment. "substantially purified" molecules are at least 60% free, preferably at least 75% free and more preferably at least 90% free of other components with which they are naturally associated. The purified nucleic acid sequence may be an isolated nucleic acid sequence.

And (3) recombination: in the context of nucleic acid molecules, the term "recombinant" refers to nucleic acid molecules produced by recombinant DNA techniques. Recombinant nucleic acid molecules may also include molecules that do not occur in nature per se, but which have been modified, altered, mutated or manipulated by man. Preferably, a "recombinant nucleic acid molecule" is a non-naturally occurring nucleic acid molecule that differs in sequence from a naturally occurring nucleic acid molecule by at least one nucleic acid. "recombinant nucleic acid molecule" may also include "recombinant constructs" comprising a series of nucleic acid molecules, preferably operably linked, that do not naturally occur in this order. Preferred methods for producing the recombinant nucleic acid molecule may include cloning techniques, directed or non-directed mutagenesis, synthesis or recombinant techniques.

Reduced expression: the expression of a nucleic acid molecule in a cell is equally used herein to "reduce" or "reduce" the expression of the nucleic acid molecule, and this means that the expression level of the nucleic acid molecule in the cell after application of the method of the invention is lower than its expression in the cell prior to the method, or lower than in a reference cell lacking a recombinant nucleic acid molecule of the invention. For example, the reference cell comprises the same construct comprising the starting regulatory nucleic acid molecule of the invention and not comprising the synthetic regulatory nucleic acid molecule of the invention. The term "reduce" or "reducing" as used herein is synonymous and means herein a reduction, preferably a significant reduction, in the expression of the nucleic acid molecule to be expressed. As used herein, "reducing" the level of a substance (e.g., a protein, mRNA, or RNA) means that the level is reduced relative to a substantially identical cell lacking a recombinant nucleic acid molecule of the invention (e.g., comprising an initial regulatory nucleic acid molecule of the invention and not comprising a synthetic regulatory nucleic acid molecule of the invention) grown under substantially identical conditions. As used herein, a "reduced" level of a substance (such as, e.g., a preRNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by a target gene) and/or a protein product encoded by a target gene means that the level is reduced by 10% or more, e.g., 20% or more, 30% or more, 40% or more, preferably 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, relative to a cell lacking a recombinant nucleic acid molecule of the invention (e.g., comprising an initial regulatory nucleic acid molecule of the invention and not comprising a synthetic regulatory nucleic acid molecule of the invention). The reduction can be determined by methods familiar to the skilled worker. Thus, the reduction in the amount of nucleic acid or protein can be determined, for example, by immunological detection of the protein. In addition, techniques such as protein assays, fluorescence, RNA hybridization, nuclease protection assays, reverse transcription (quantitative RT-PCR), ELISA (enzyme-linked immunosorbent assay), western blotting, radioimmunoassay (RIA) or other immunoassays and fluorescence activated cell analysis (FACS) can be used to measure specific proteins or RNA in cells. Depending on the type of protein product that has been reduced, its activity or effect on the phenotype of the organism or cell may also be determined. Methods for determining the amount of protein are known to the skilled person. Examples which may be mentioned are: the microscale Biuret method (Goa J (1953) Scand J Clin Lab Invest 5.

A sense: the term "sense" is understood to mean a nucleic acid molecule having a sequence which is complementary to or identical to a target sequence, for example a sequence which binds to a protein transcription factor and is involved in the expression of a given gene. According to a preferred embodiment, the nucleic acid molecule comprises a gene of interest and elements allowing the expression of the gene of interest.

Significant increase or decrease: an increase or decrease greater than the margin of error inherent in the measurement technique, for example in enzyme activity or in gene expression, preferably an increase or decrease of about 2-fold or more in the activity of a control enzyme or in expression in a control cell, more preferably an increase or decrease of about 5-fold or more, and most preferably an increase or decrease of about 10-fold or more.

Small nucleic acid molecules: by "small nucleic acid molecule" is understood a molecule consisting of a nucleic acid or a derivative thereof, such as RNA or DNA. They may be double-stranded or single-stranded and have a length of between about 15 and about 30bp, such as between 15 and 30bp, more preferably between about 19 and about 26bp, such as between 19 and 26bp, even more preferably between about 20 and about 25bp, such as between 20 and 25 bp. In a particularly preferred embodiment, the oligonucleotide is between about 21 and about 24bp in length, for example between 21 and 24bp. In a most preferred embodiment, the small nucleic acid molecules are about 21bp and about 24bp in length, e.g., 21bp and 24bp.

Substantially complementary: in the broadest sense, the term "substantially complementary" as used herein in reference to a nucleotide sequence relative to a reference or target nucleotide sequence means a nucleotide sequence having a percentage of identity (the latter being equivalent to the term "identical" in this context) of at least 60%, more desirably at least 70%, more desirably at least 80% or 85%, preferably at least 90%, more preferably at least 93%, still more preferably at least 95% or 96%, still more preferably at least 97% or 98%, still more preferably at least 99% or most preferably 100% between the substantially complementary nucleotide sequence and the fully complementary sequence of said reference or target nucleotide sequence. Preferably, the identity is assessed for the reference nucleotide sequence over a length of at least 19 nucleotides, preferably at least 50 nucleotides, more preferably the entire length of the nucleic acid sequence (if not, as further explained below). Sequence comparisons were performed based on the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J mol. Biol.48:443-453; as defined above) using the default GAP analysis of university of Wisconsin GCG, SEQWEB application of GAP. A nucleotide sequence that is "substantially complementary" to a reference nucleotide sequence hybridizes to the reference nucleotide sequence under low stringency conditions, preferably medium stringency conditions, most preferably high stringency conditions (as defined above).

As used herein, "target region" means a region that is nearly, for example, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 60 bases, 70 bases, 80 bases, 90 bases, 100 bases, 125 bases, 150 bases, 200 bases, or 500 bases or more from a target site or comprises a target site where a sequence of a donor DNA molecule is introduced into the genome of a cell.

"target site" as used herein means a location in the genome where a double-stranded break or one or a pair of single-stranded breaks (nicks) is induced using recombinant techniques such as Zn finger, TALENs, restriction enzymes, homing endonucleases, RNA-guided nucleases, RNA-guided nickases such as CRISPR/Cas nucleases or nickases, etc.

And (3) transgenosis: the term "transgene" as used herein refers to any nucleic acid sequence that is introduced into the genome of a cell by experimental manipulation. A transgene may be an "endogenous DNA sequence" or a "heterologous DNA sequence" (i.e., "foreign DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence that is naturally present in a cell into which it is introduced, so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence.

Transgenic: the term "transgenic" when referring to an organism means the transformation, preferably stable transformation, of an organism with a recombinant DNA molecule, preferably comprising a suitable promoter operably linked to a DNA sequence of interest.

Carrier: the term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a genomic integrative vector, or "integrative vector", which can integrate into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, i.e., a nucleic acid molecule capable of undergoing extrachromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In the present specification, "plasmid" and "vector" are used interchangeably unless not the case from the context. Expression vectors designed to produce RNA as described herein in vitro or in vivo may contain sequences recognized by any RNA polymerase, including mitochondrial RNA polymerase, RNA pol I, RNA pol II, and RNA pol III. These vectors can be used to transcribe a desired RNA molecule in a cell according to the invention.

Brief Description of Drawings

FIG. 1 is a schematic view of a

A plasmid map of a single CRISPR/Cas9 plasmid pCC009 is described. Plasmid pCC009 is a derivative of plasmid pJOE8999.1, which carries the spacer of the B.licheniformis amyB gene and the DNA donor sequences HomA and HomB 5 'and 3' respectively of the amyB gene. PmanP: the promoter of the bacillus subtilis manP gene; pUC ORI: coli high copy replication origin; kanamycin resistance gene that is functional in both bacillus and escherichia coli; rep pE194: a fragment of plasmid pE194 that confers replication of a temperature-sensitive plasmid in bacillus; pvanP: a promoter that drives expression of the spacer region, sgRNA (crRNA repeat +' gRNA); a T0 terminator from λ; t1t2 terminator from e.coli rrnB gene; homA and HomB: 5 'and 3' sequences of the amyB gene fused together for gene deletion; cas9: cas9 endonuclease from streptococcus pyogenes(s).

FIG. 2 is a schematic diagram:

sequence alignment of selected regions of mutated promoter sequences-nt 15 to nt.128 reference against promoter sequences PV4 (SEQ ID 028) and PV8 (SEQ ID 029) are shown. The-35 and-10 regions, the Transcription Start Site (TSS) and the Shine Dalgarno Sequence (SD) are depicted in italic letters and shaded in grey inside the reference promoter sequences of the PV4 (SEQ ID 028) and PV8 (SEQ ID 029) promoters. Nucleotide deletions, insertions and mutations are depicted in bold.

FIG. 3

Single colonies were analyzed by colony PCR for bacillus licheniformis amyB gene deletion with oligonucleotides SEQ ID 009 and SEQ ID010 located outside the region of homology for gene deletion. The gene deletion efficiency of the bacillus licheniformis amylase amyB gene (as a percentage of clones with inactivated amylase gene relative to the total of 20 clones analyzed for each gene deletion construct) was plotted for each gene deletion construct as shown. A. The relative deletion efficiency of deletion plasmids derived from PV4 promoter variants is described. B. The relative deletion efficiency of deletion plasmids derived from PV8 promoter variants is described.

FIG. 4

A. The gene deletion efficiency of bacillus licheniformis hag gene (as a percentage of clones with inactivated hag gene relative to a total of 20 analyzed clones) was plotted against the two deletion constructs and promoter variants as shown, respectively. The mean of three independent experiments is shown along with the standard deviation. The hag gene deletion was analyzed by colony PCR using oligonucleotides SEQ ID 087 and SEQ ID 088 located outside the region of homology for gene deletion. B. The relative mutation efficiencies of the two deletion constructs and the promoter variant, respectively, for introducing point mutations within the bacillus licheniformis degU gene are described as the percentage of clones with mutated degU gene relative to the total of 20 analyzed clones. The mean of three independent experiments is shown along with the standard deviation. The gene mutation of the degU gene was analyzed by colony PCR with the oligonucleotides SEQ ID 089 and SEQ ID 090 located outside the region of homology used to introduce the gene mutation, followed by PstI restriction digestion of the PCR fragment to distinguish between the native and mutant degU loci.

FIG. 5

A. The gene deletion efficiency of the bacillus subtilis amylase amyE gene (as a percentage of clones with inactivated amyE gene relative to the total of 20 analyzed clones) was plotted against the two deletion constructs and promoter variants, respectively, as shown. The mean of three independent experiments is shown along with the standard deviation. Gene deletion of the amyE gene was analyzed by colony PCR with oligonucleotides SEQ ID 091 and SEQ ID 092 located outside the homology region for gene deletion. B. The relative deletion efficiencies of the two deletion constructs and promoter variants, respectively, for deleting the subtilisin aprE gene of bacillus subtilis are described as the percentage of clones with inactivated aprE gene relative to the total of 20 analyzed clones. The mean of three independent experiments is shown along with the standard deviation. The aprE gene was analyzed for gene deletion by colony PCR using oligonucleotides SEQ ID 093 and SEQ ID 094 located outside the homology region for gene deletion.

FIG. 6

A. The gene deletion efficiency of the bacillus licheniformis vpr gene (as a percentage of clones with inactivated vpr gene relative to the total of 20 analyzed clones) was plotted against the three deletion constructs and spacer variants, respectively, as shown. The gene deletion of the vpr gene was analyzed by colony PCR with oligonucleotides SEQ ID 095 and SEQ ID 096 located outside the homology region for gene deletion. B. The relative deletion efficiencies of the three deletion constructs and the spacer variant, respectively, for deleting the epr gene from bacillus licheniformis are described as a percentage of clones with inactivated epr gene relative to a total of 20 analyzed clones. The gene deletion of the epr gene was analyzed by colony PCR using the oligonucleotides SEQ ID 097 and SEQ ID 098 located outside the homology region for gene deletion.

FIG. 7

The gene integration efficiency of the PaprE-GFPmut2 expression cassette in place of the Bacillus licheniformis amyB gene (as a percentage of clones with integrated PaprE-GFPmut2 expression cassette over the total of 20 analyzed clones) was plotted for two different Bacillus licheniformis strains Bli #005 and P308, respectively, as indicated. The mean of two independent experiments is shown along with the standard deviation. Integration was analyzed by colony PCR with oligonucleotides SEQ ID 009 and SEQ ID010 located outside the region of homology for gene integration.

FIG. 8

The gene deletion efficiencies for the sporulation genes sigE, sigF and spoIIE of Bacillus pumilus, as a percentage of clones with inactivated sporulation genes relative to the total of 20 clones for each analyzed sporulation gene, were plotted as indicated. The gene deletions of the sigE, sigF and spoIIE genes were analyzed by colony PCR with oligonucleotides SEQ ID099 and SEQ ID 100, SEQ ID 101and SEQ ID 102 and SEQ ID 103and SEQ ID 104, respectively, which lie outside the homology regions for gene deletion.

Examples

Materials and methods

The following examples serve only to illustrate the invention. Numerous possible variations, which will be apparent to those skilled in the art, also fall within the scope of the invention.

Unless otherwise stated, the following experiments have been performed by applying standard equipment, methods, chemicals and biochemicals as used in genetic engineering and fermentative production of chemical compounds by culturing microorganisms. See also Sambrook et al (Sambrook, J. And Russell, D.W. molecular cloning. A Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y.) and Chmiel et al (bioprocess Stechnik 1. Einf. Huning in die Bioverfahrentechnik, gustav Fischer Verlag, stuttgart, 1991).

Electrocompetent Bacillus licheniformis cells and electroporation

The DNA was transformed into Bacillus licheniformis strains DSM641 and ATCC53926 by electroporation. Preparation and DNA transformation of electrocompetent Bacillus licheniformis cells was performed essentially as described by Brigidi et al (Brigidi, P., mateuzzi, D. (1991). Biotechnol. Techniques 5,5), but withThe following modifications: once the DNA was transformed, the cells were recovered in 1ml LBSPG buffer and incubated at 37 ℃ for 60 minutes (

J.,1989, fems microbio.lett., 61), and thereafter spread on selective LB agar plates.

To overcome the restriction modification system specific for B.licheniformis of B.licheniformis strains DSM641 and ATCC53926, plasmid DNA was isolated from Ec #098 cells as described below. For transfer into the bacillus licheniformis restriction enzyme knockout strain, plasmid DNA was isolated from e.coli INV110 cells (Life technologies).

Electrocompetent Bacillus pumilus cells and electroporation

The DNA was transformed into Bacillus pumilus DSM14395 by electroporation. Preparation and DNA transformation of electrocompetent Bacillus pumilus DSM14395 cells were performed as described for Bacillus licheniformis cells.

To overcome the restriction modification system specific for B.pumilus, plasmid DNA was isolated from E.coli DH10B cells and methylated in vitro with the whole cell extract from B.pumilus DSM14395 according to the method described for B.licheniformis in DE 4005025.

Electrocompetent Bacillus subtilis cells and electroporation

The DNA was transformed into Bacillus subtilis ATCC6051a by electroporation as described for Bacillus licheniformis and Bacillus pumilus, respectively. Plasmid DNA isolated from E.coli DH10B cells can be readily used for transfer into Bacillus subtilis.

Plasmid isolation

Plasmid DNA was isolated from Bacillus and E.coli cells by standard molecular biology methods described by (Sambrook, J. And Russell, D.W. "molecular cloning Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY.2001) or by alkaline lysis methods (Birnboim, H.C., doly, J. (1979) Nucleic Acids Res 7 (6): 1513-1523). In contrast to E.coli, bacillus cells were treated with 10mg/ml lysozyme for 30 minutes at 37 ℃ before lysing the cells.

The oligonucleotide is annealed to form an oligonucleotide-duplex.

The oligonucleotides were adjusted to a concentration of 100. Mu.M in water. Mu.l of the forward oligonucleotide and 5. Mu.l of the corresponding reverse oligonucleotide were added to 90. Mu.l of 30mM Hepes-buffer (pH 7.8). The reaction mixture was heated to 95 ℃ for 5 minutes, followed by annealing with a temperature ramp of 95 ℃ to 4 ℃ at a reduced temperature of 0.1 ℃/sec (Cobb, r.e., wang, y, and Zhao, h. (2015). High-Efficiency Multiplex Genome Editing of Streptomyces specificities Using an Engineered CRISPR/Cas System. ACS Synthetic Biology,4 (6), 723-728).

Molecular biological methods and techniques

Standard methods in molecular biology, not limited to culturing Bacillus and E.coli microorganisms, DNA electroporation, isolating genomic and plasmid DNA, PCR reactions, cloning techniques, are essentially performed as described in Sambrook and Russell (Sambrook, J.and Russell, D.W.; molecular cloning Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. 2001).

Bacterial strains

Escherichia coli strain Ec #098

Coli strain Ec #098 is an E.coli INV110 strain (Life technologies) carrying the expression plasmid pMDS003 encoding a DNA methyltransferase (WO 2019016051).

Production of Bacillus licheniformis Gene k.o Strain

For gene deletion in Bacillus licheniformis strains DSM641 and ATCC53926 (US 5352604) and derivatives thereof, the deletion plasmids were transformed into E.coli strain Ec #098 which was made competent according to the Chung method (Chung, C.T., niemela, S.L. and Miller, R.H. (1989) One-step preparation of the compatible Escherichia coli: transformation and storage of bacterial cells in the same solution.) Proc.Natl.Acad.Sci.U.S.A 86,2172-2175 followed by selection on agar plates containing 100. Mu.g/ml ampicillin and 30. Mu.g/ml chloramphenicol at 37 ℃. Plasmid DNA was isolated from independent clones and used for subsequent transformation into a Bacillus licheniformis strain. The isolated plasmid DNA carries the DNA methylation patterns of Bacillus licheniformis strains DSM641 and ATCC53926, respectively, and is protected from degradation when transferred into Bacillus licheniformis.

Bacillus licheniformis P304: deletion of restriction endonucleases

Electrocompetent Bacillus licheniformis DSM641 cells (US 5352604) were prepared as described above and the plasmid was deleted with 1. Mu.g of pDel006 restriction enzyme gene isolated from E.coli Ec #098 and subsequently plated on LB agar plates containing 5. Mu.g/ml erythromycin at 30 ℃.

The gene deletion process was performed as follows:

the plasmid-carrying Bacillus licheniformis cells were grown at 45 ℃ on LB agar plates containing 5. Mu.g/ml erythromycin to drive integration of the deleted plasmid into the chromosome by means of Campbell recombination process, while one of the homologous regions of pDel006 is homologous to the sequence of either 5 'or 3' of the aprE gene. Clones were picked and incubated at 45 ℃ for 6 hours in LB medium without selective pressure, followed by plating on LB agar plates containing 5. Mu.g/ml erythromycin at 30 ℃. Independent grams were picked and screened for successful genome deletions of the restriction enzyme gene by colony PCR analysis with oligonucleotides SEQ ID 014 and SEQ ID 015. Putative deletion positive independent clones were picked and subjected to two consecutive overnight incubations at 45 ℃ in LB medium without antibiotics to eliminate plasmids and plated on LB agar plates overnight at 37 ℃. Single clones were analyzed by colony PCR for successful genome deletion of the restriction enzyme gene. An erythromycin sensitive single clone correctly deleted of the restriction enzyme gene was isolated and designated Bacillus licheniformis P304.

Bacillus licheniformis P308: deletion of poly-gamma-glutamic acid synthetic gene

Electrocompetent Bacillus licheniformis P304 cells were prepared as described above and transformed with 1. Mu.g of pDel007 pga gene deletion plasmid isolated from E.coli INV110 cells (Life technologies) and subsequently plated on LB agar plates containing 5. Mu.g/ml erythromycin at 30 ℃.

The gene deletion process was performed as described for the deletion of the restriction enzyme gene.

The deletion of the pga gene was analyzed by PCR with the oligonucleotides SEQ ID 017 and SEQ ID 018. The resulting B.licheniformis strain with the deletion of the pga synthesis gene was named B.licheniformis P308.

Bacillus licheniformis Bli #002: deletion of aprE Gene

Electrocompetent B.licheniformis ATCC53926 cells were prepared as described above and the plasmid was deleted with 1. Mu.g of pDel003 aprE gene isolated from E.coli Ec #098 and subsequently plated on LB agar plates containing 5. Mu.g/ml erythromycin at 30 ℃.

The gene deletion process was performed as described for the deletion of the restriction enzyme gene. Deletion of the aprE gene was analyzed by PCR with oligonucleotides SEQ ID 020 and SEQ ID 021. The resulting B.licheniformis strain with the aprE gene deleted was designated Bli #002.

B.licheniformis Bli #005: deletion of poly-gamma-glutamic acid synthetic gene

In B.licheniformis Bli #002 the poly-gamma-glutamate synthesis gene was deleted as described for the deletion of the pga gene in B.licheniformis P304, with the difference that the pDel007 plasmid was isolated from E.coli Ec #098 cells. The resulting strain was named Bli #005.

Plasmids

pEC194 RS-Bacillus temperature sensitive deletion plasmid.

Plasmid pE194 was PCR amplified with oligonucleotides SEQ ID 001 and SEQ ID 002 flanking the PvuII site, digested with restriction endonuclease PvuII and ligated into the restriction enzyme SmaI digested vector pCE 1. pCE1 is a pUC18 derivative in which the BsaI site inside the ampicillin resistance gene has been removed by silent mutation. The ligation mixture was transformed into E.coli DH10B cells (Life technologies). The transformants were spread and incubated overnight at 37 ℃ on LB agar plates containing 100. Mu.g/ml ampicillin. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion. The resulting plasmid was designated pEC194S.

Type II assembly mRFP cassettes comprising an additional nucleotide restriction site BamHI were PCR amplified from plasmid pBSd141R (accession number: KY 995200) (radiack, J., meyer, D., lautenscheler, N. And Mascher, T.2017.Bacillus SEVA sites: A Golden Gate-based toolkit to create a personalized integration vector for Bacillus subtilis) Sci.Rep.7:14134 with oligonucleotides SEQ ID 003 and SEQ ID 004. The PCR fragment and pEC194S were restricted with the restriction enzyme BamHI, followed by ligation and transformation into E.coli DH10B cells (Life technologies). The transformants were spread and incubated overnight at 37 ℃ on LB agar plates containing 100. Mu.g/ml ampicillin. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion. The resulting plasmid pEC194RS carries an mRFP cassette with an open reading frame aligned with the open reading frame of the erythromycin resistance gene.

pDel003-aprE gene deletion plasmid

A gene deletion plasmid for the B.licheniformis aprE gene was constructed with plasmid pEC194RS and the gene synthesis construct SEQ ID 019 containing the genomic region 5 'and 3' of the aprE gene flanked by BsaI sites compatible with pEC194 RS. Type II assembly with the restriction endonuclease BsaI was performed as described (radius, J., meyer, D., lautenscheler, N., and Mascher, T.2017.Bacillus SEVA sites: A Golden Gate-based toolkit to create a personalized integration vector for Bacillus subtilis, bacillus SEVA siblings: gold Gate-based toolkit for Bacillus subtilis.) Sci.Rep.7:14134 and the reaction mixture was subsequently transformed into E.coli DH10B cells (Life technologies). The transformants were spread and incubated overnight at 37 ℃ on LB agar plates containing 100. Mu.g/ml ampicillin. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion. The resulting aprE deletion plasmid was designated pDel003.

pDel 006-restriction enzyme gene deletion plasmid

The gene deletion plasmid for the restriction enzyme gene (SEQ ID 012) in the bacillus licheniformis DSM641 restriction modification system (SEQ ID 011) was constructed with plasmid pEC194RS and the gene synthesis construct SEQ ID 013 comprising the genomic region of the restriction enzyme genes 5 'and 3' flanked by a BsaI site compatible with pEC194 RS. Type II assembly with restriction endonuclease BsaI was performed as described above and the reaction mixture was subsequently transformed into e.coli DH10B cells (Life technologies). The transformants were spread and incubated overnight at 37 ℃ on LB agar plates containing 100. Mu.g/ml ampicillin. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion. The resulting restriction enzyme deletion plasmid was designated pDel006.

pDel 007-poly-gamma-glutamic acid synthetic gene deletion plasmid

Deletion plasmids for deleting the genes involved in the production of poly-gamma-glutamate (pga) of bacillus licheniformis, i.e. ywsC (pgsB), ywtA (pgsC), ywtB (pgsA), ywtC (pgsE), were generated as described in pDel006, however using the gene synthesis construct SEQ ID 016 comprising genomic regions flanked on the 5 'and 3' sides of the ywsC, ywtA (pgsC), ywtB (pgsA), ywtC (pgsE) genes, flanked by BsaI sites flanking pEC194 RS. The resulting pga deletion plasmid was designated pDel007.

Plasmid p689-T2A-lac

Plasmid p689-T2A-lac contains the lacZ-alpha gene flanked by BpiI restriction sites, which in turn are flanked 5 'by the T1 terminator of the E.coli rrnB gene and 3' by the T0 lambda terminator, and ordered as a gene synthesis construct (SEQ ID 073).

Plasmid p890 PaprE-GFPmut2

The promoter of the Bacillus licheniformis aprE gene of plasmid pCB56C (US 5352604) was PCR amplified with oligonucleotides SEQ ID074 and SEQ ID 075. The GFPmut2 gene variant with flanking BpiI restriction site (accession No. AF 302837) (SEQ ID 076) was ordered as a gene synthesis fragment (Geneart renberg). The gene expression construct comprising the PaprE promoter of Bacillus licheniformis origin fused to the GFPmut2 variant was cloned into the plasmid p689-T2A-lac by type II assembly with the restriction endonuclease BpiI as described (radius, J., meyer, D., lautenscheler, N. And Mascher, T.2017.Bacillus SEVA sites: A gold Gate-based toolkit to generate a personalized integration vector for Bacillus subtilis.) Sci.Rep.7:14134 and the reaction mixture was subsequently transformed into electro-competent E.coli DH10B cells. The transformants were spread and incubated overnight at 37 ℃ on LB agar plates containing 100. Mu.g/ml ampicillin. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion and sequenced. The resulting plasmid was designated p890 PaprE-GFPmut2.

Plasmid pJOE8999.1:

altenbuchner j.2016.Editing of the Bacillus subtilis genome by the CRISPR-Cas9system (editing of the Bacillus subtilis genome by the CRISPR-Cas9 system.) Appl Environ Microbiol82:5421-5.

Plasmid pJOE-T2A

To allow one-step cloning of sgrnas and homology regions for DSB repair based on type II assembly (T2A), the CRISPR/Cas9 plasmid pjoe8889.1 was modified as follows. Type II assembly mRFP cassettes from plasmid pBSd141R (accession number: KY 995200) (radiack, J., meyer, D., lautenscheler, N. And Mascher, T.2017.Bacillus SEVA sites: A Golden Gate-based toolkit to generate personalized integration vectors for Bacillus subtilis.) Sci.Rep.7:14134 were so modified as to remove multiple restriction sites and BpiI restriction sites and ordered as gene synthesis flanking fragments with SfiI restriction sites (SEQ ID 005). The plasmid was designated p #732. Plasmid p #732 and plasmid pJOE8999.1 were digested with SfiI (New England Biolabs, NEB) and the mRFP cassette of p #732 was ligated into SfiI-digested pJOE8999.1, followed by transformation into competent E.coli DH10B cells. Purple colonies of positive clones were screened on LB agar plates containing IPTG/X-Gal and kanamycin (20. Mu.g/ml) (blue-white screening and mRFP1 expression). The resulting sequence-verified plasmid was designated pJOE-T2A.

Plasmid pBW732

The 5 'homology region (also referred to as HomA) and the 3' homology region (also referred to as HomB) adjacent to the amylase amyB gene of Bacillus licheniformis DSM641 were ordered as synthetic gene synthesis fragments with flanking XmaI restriction sites (SEQ ID 006). Plasmid pJOE8999.1 and the synthetic amyB-HomAB fragment were cleaved with the restriction endonuclease XmaI, followed by ligation with T4-DNA ligase (NEB) and transformation into electrocompetent E.coli DH10B cells. The correct plasmid was recovered and designated pBW732.

Plasmid pBW742

Geneius 11.1.5 (https:// www.geneious.com) was used to design a 20bp target sequence for the amyB gene of the sgRNA. The resulting 5' phosphorylated oligonucleotides SEQ ID 007 and SEQ ID 008 were renatured to form an oligonucleotide duplex. Type II assembly by using the restriction endonuclease BsaI as described (radial, J., meyer, D., lautenscheler, N., and Mascher, T.2017.Bacillus SEVA sites: A Golden Gate-based toolkit to create a personalized integration vector for Bacillus subtilis, bacillus SEVA siblings: gold Gate-based toolkit for producing a personalized integration vector for Bacillus subtilis) Sci.Rep.7: 14134) was used for the following components: pBW732 and oligonucleotide duplexes (SEQ ID 007, SEQ ID 008) construct a CRISPR/Cas 9-based gene deletion plasmid for the Bacillus licheniformis amyB gene. The reaction mixture was transformed into E.coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion and sequenced. The resulting amyB deletion plasmid was designated pBW742.

T2A CRISPR objective vectors pCC027 and pCC028

Plasmids pCC014 and pCC025 were modified such that the region covering the spacer-sgRNA and the amyB gene flanked by homologous regions was replaced by the T2A cassette from plasmid pJOE-T2A. The backbone of pCC014 and pCC025 was PCR amplified with oligonucleotides SEQ ID 050 and SEQ ID 051 and the T2A assembly cassette was PCR amplified from pJOE-T2A with oligonucleotides SEQ ID 048 and SEQ ID 049, followed by PCR purification using the High Pure PCR purification kit, digestion with DpnI and gel purification. The corresponding backbone PCR fragment and T2A cassette PCR fragment were renatured in a 10. Mu.l Gibson reaction and subsequently transformed into E.coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion and sequenced. The resulting pCC 014-derived and pCC 025-derived T2A plasmid derivatives were designated pCC027 and pCC028, respectively.

pCC029-hag gene deletion plasmid

The hag gene 20bp target sequence for sgRNA was designed using Geneious 11.1.5 as described previously. The 5' phosphorylated resulting oligonucleotides SEQ ID 056 and SEQ ID 057 were annealed to form the oligonucleotide duplex described above. The genomic region of hag gene 5 'and 3' was PCR amplified on the genomic DNA of B.licheniformis DSM641 using oligonucleotides SEQ ID 054 and SEQ ID 053 and SEQ ID 052 and SEQ ID 55, followed by fusion with the flanking oligonucleotides SEQ ID 053 and SEQ ID 054 by overlap extension PCR. The resulting PCR product was column purified (Qiagen PCR purification kit). The type II assembly by using the restriction endonuclease BsaI was used as described above for the following components: plasmid pCC027 (PV 4-5 promoter variant), hag gene with flanking BsaI restriction site fused to homology region and oligonucleotide duplexes (SEQ ID 056, SEQ ID 057) a CRISPR/Cas 9-based gene deletion plasmid was constructed against the B.licheniformis hag gene. The reaction mixture was transformed into E.coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion and sequenced. The resulting hag gene deletion plasmid was designated pCC029.

pCC030-hag gene deletion plasmid

The hag gene deletion construct was constructed as pCC029, however plasmid pCC028 (PV 8-7 promoter variant) was used.

pCC031-degU32 gene editing plasmid

The degU32 genome editing construct was constructed as for pCC029 to introduce the degU H12L mutation, with the following modifications.

Mutations were introduced for the degU H12L mutation and silent point mutations were introduced to remove the degU32 homology region of the PAM site as a gene synthesis construct (SEQ ID 058) with flanking BsaI sites ordered (Geneart, rengen burgh). The 20bp target sequence for the degU gene of the sgRNA was designed as described previously and the resulting oligonucleotides SEQ ID 059 and SEQ ID 060 phosphorylated at 5' were renatured to form oligonucleotide duplexes.

pCC032-degU32 gene editing plasmid

The degU32 genome editing construct was generated as described for pCC031, however plasmid pCC028 (PV 8-7 promoter variant) was used.

pCC033-amyE gene deletion plasmid

Fragments comprising amyE spacer-sgRNA and homologous regions of the 5 'and 3' regions of the Bacillus subtilis amyE gene were PCR amplified from plasmid pCC004 (WO 17186550) using oligonucleotides SEQ ID 061 and SEQ ID 062 flanking BsaI restriction sites. A gene deletion plasmid for the amylase amyE gene based on CRISPR/Cas9 was then constructed as described above with plasmid pCC027 (PV 4-5 promoter variant) and PCR amplified fragments by type II assembly with restriction endonuclease BsaI. The reaction mixture was transformed into E.coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion and sequenced. The resulting amyE gene deleted plasmid was designated pCC033.

pCC034-amyE gene deletion plasmid

The amyE gene deletion construct was constructed as pCC033, however plasmid pCC028 (PV 8-7 promoter variant) was used.

pCC035-aprE gene deletion plasmid

Fragments comprising the aprE spacer (SEQ ID 064) -sgRNA and the homologous regions of the 5 'and 3' regions of the B.subtilis aprE gene were ordered as synthetic gene fragments with flanking BsaI restriction sites (SEQ ID 063). A CRISPR/Cas 9-based gene deletion plasmid for the protease aprE gene was then constructed as described above with plasmid pCC027 (PV 4-5 promoter variant) and the gene synthesis construct by type II assembly with the restriction endonuclease BsaI. The reaction mixture was transformed into E.coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion and sequenced. The resulting aprE gene deletion plasmid was designated pCC035.

pCC036-aprE gene deletion plasmid

The aprE gene deletion construct was constructed as pCC035, however plasmid pCC028 (PV 8-7 promoter variant) was used.

pCC037-pCC039-vpr gene deletion plasmid

CRISPR/Cas9 gene deletion constructs pCC037, pCC038 and pCC039 for the bacillus licheniformis protease vpr gene were constructed as described for pCC035, however with a synthetic gene fragment (SEQ ID 065) comprising the vpr spacer-sgRNA and homologous regions of the 5 'and 3' regions of the vpr gene. The resulting plasmids pCC037, pCC038 and pCC039 differed in the vpr spacer sequence within SEQ ID 065 (SEQ ID 066, SEQ ID 067, SEQ ID 068).

pCC040-pCC042-epr gene deletion plasmid

CRISPR/Cas9 gene deletion constructs pCC040, pCC041 and pCC042 of the bacillus licheniformis protease epr gene were constructed as described for pCC035, but with a synthetic gene fragment (SEQ ID 069) comprising the epr spacer-sgRNA and homologous regions of the 5 'and 3' regions of the epr gene. The resulting plasmids pCC040, pCC041 and pCC042 differ in the vpr spacer sequence within SEQ ID 069 (SEQ ID 070, SEQ ID 071, SEQ ID 072).

pCC043-GFP gene integration plasmid

The amyB gene 20bp target sequence for sgRNA was ordered as 5' phosphorylated oligonucleotides SEQ ID 007 and SEQ ID 008, followed by renaturation to form an oligonucleotide duplex. The 5 'and 3' regions of the B.licheniformis amyB gene were PCR amplified with oligonucleotides SEQ ID 077 and SEQ ID 078 and SEQ ID 079 and SEQ ID 080 respectively.

The type II assembly by using the restriction endonuclease BsaI was used as described above for the following components: pCC027, oligonucleotide duplexes (SEQ ID 007, SEQ ID 008), a PCR fragment of the 5 'homology region of the amyB gene, p890-PaprE-GFPmut2 and a PCR fragment of the 3' homology region of the amyB gene construct a CRISPR/Cas 9-based gene integration plasmid construct replacing the Bacillus licheniformis amyB gene. The reaction mixture was transformed into E.coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion and sequenced. The resulting gene integration plasmid based on CRISPR/Cas9 was named pCC043.

pCC 044-Bacillus pumilus sigE gene deletion plasmid

CRISPR/Cas9 gene deletion construct pCC044 of the sigE gene of bacillus pumilus DSM14395 was constructed as described for pCC035, however with a synthetic gene fragment (SEQ ID 082) comprising the sigE spacer (SEQ ID 081) -sgRNA and homologous regions of the 5 'and 3' regions of the sigE gene.

pCC 045-Bacillus pumilus sigF gene deletion plasmid

CRISPR/Cas9 gene deletion construct pCC045 of the sigF gene of bacillus pumilus DSM14395 was constructed as described for pCC035, however with a synthetic gene fragment (SEQ ID 084) comprising the sigF spacer (SEQ ID 083) -sgRNA and homologous regions of the 5 'and 3' regions of the sigF gene.

pCC 046-Bacillus pumilus spoIIE gene deletion plasmid

CRISPR/Cas9 gene deletion construct pCC046 of spolie gene of bacillus pumilus DSM14395 was constructed as described for pCC035, however with a synthetic gene fragment (SEQ ID 086) comprising the spolie spacer (SEQ ID 085) -sgRNA and homologous regions of the 5 'and 3' regions of the spolie gene.

Example 1: construction of CRISPR/Cas9 genome editing plasmids carrying constitutive promoters to introduce a constitutive promoter driving Cas9 enzyme expression in plasmid pBW742, a two-step approach was employed.

First, the t1t2t0 terminator (derived from pMUTIN) was introduced 5' to the promoter PmanP of pBW742 to prevent potential read-through from the kanamycin selectable marker.

Assembled by Gibson (

HiFi DNA assembly cloning kit, new England Biolabs) so that the terminator sequence t1t2t0 is integrated into pBW742 upstream of the mannose promoter. For this purpose, the terminator fragment (0.44 kb) was amplified by PCR using pMutin2 (accession number AF 072806) as template, with oligonucleotides SEQ ID 024 and SEQ ID 025. The corresponding vector backbone of pBW742 was amplified with oligonucleotides SEQ ID 022 and SEQ ID 023. The pBW742 amplicon was purified using PCR product purification kit (Roche). After subsequent digestion of the pBW742 PCR product with DpnI (New England Biolabs), both PCR fragments were gel purified using Qia a Rapid gel extraction kit (Qiagen, hilden, germany) and renatured at 50 ℃ for 1 hour at a rate of 1:2. Coli strain DH10B with the assembly reaction transformation, then shop in containing 20 u g/ml kanamycin LB agar plate. Plasmid DNA was isolated from independent clones and analyzed for accuracy by restriction digestion and sequenced.

There was a deviation from the published pMutin2 reference sequence. SEQ ID 026 covers a portion of the sequence of pMutin2 and SEQ ID 027 covers the sequence deviations present in the corresponding region of pMutin2 present in the resulting plasmid pCC 009.

Secondly, the mannose-inducible promoter PmanP is exchanged for two promoter variants of the constitutive promoter Pveg of Bacillus subtilis-namely PV4 and PV8 (Guizou, S., V.Sauveplane, H.J.Chang, C.Clert, N.Declerck, M.Jules and J.Bonnet.2016.A part of the genomic expression in Bacillus subtilis). These promoter variants comprise a Pveg promoter derived from an adapted Pveg promoter library, a standardized TSS (transcription start site) region and a standardized ribosome binding site region R0, wherein the promoter library is screened for single copy levels in bacillus subtilis relative to the altered expression level of the promoter variant. The promoter sequences PV4 and PV8 are listed as SEQ ID 028 and SEQ ID 029, respectively.

Integration of both promoter variants was performed by Gibson assembly. The PV4 and PV8 fragments were amplified stepwise. For the promoter fragment, using pCC009 as template, oligonucleotides SEQ ID 024 and SEQ ID 030 were used for the first PCR (Phusion hi-fi DNA polymerase-NEB) and the resulting product served as template for the second PCR, oligonucleotides SEQ ID 024 and SEQ ID 031 directed against PV4 and SEQ ID 024 and SEQ ID 033 directed against PV8.

The vector backbone of pCC009 was PCR amplified using oligonucleotides SEQ ID 022 and SEQ ID 032. After purification of the vector amplicons with the PCR purification kit (Roche), the PCR products were digested with DpnI to remove the remaining circular plasmid DNA from the PCR reaction. Subsequently, the digested vector and the two promoter fragments were purified using the Qiaquick gel extraction kit (Qiagen, hilden, germany). Vector amplicon pCC009 was then renatured with promoter fragments PV4 and PV8, respectively, thus replacing the mannose promoter PmanP with the PV4 and PV8 variants of the Pveg promoter.

The renaturation reactions were subsequently transformed into E.coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃. Plasmid DNA was isolated from 9 individual clones of the PV4 promoter and from 8 independent clones derived from the promoter variant PV8 and analyzed for accuracy by sequencing.

Table 1 summarizes the sequencing results for the various promoter variants:

analysis of clones from the PV 4-cloning reaction revealed that only sequences with point mutations, nucleotide insertions or deletions within the PV4 region could be recovered.

Analysis of clones from the PV 8-cloning reaction revealed that only sequences with point mutations, nucleotide insertions or deletions within the PV8 region could be recovered. The resulting plasmids are summarized in table 1.

TABLE 1

Gene deletion efficiency of CRISPR/Cas 9-based deletion plasmid

Electrocompetent bacillus licheniformis P308 cells were prepared as described above and transformed with 1 μ g of the amyB deletion plasmids pCC010-012, pCC014-017, pCC019-026 (with different promoter variants as described in table 1) isolated from e.coli INV110 cells (Life technologies), followed by plating on LB agar plates containing 20 μ g/ml kanamycin and incubation at 37 ℃ overnight.

The following day, 20 clones of each transformation reaction were subjected to colony PCR with oligonucleotides SEQ ID 009 and SEQ ID010 to analyze successful deletion of the amyB gene based on CRISPR/Cas9 and further transferred onto fresh LB agar plates without antibiotics followed by overnight incubation at 48 ℃ for plasmid elimination.

The amyB gene deletion efficiency of each CRISPR/Cas 9-based deletion plasmid was calculated as the ratio of successful gene deletions in percent relative to the total number of analyzed clones based on the appearance of the expected smaller specific PCR amplicon compared to the larger specific PCR amplicon of the wild-type amyB locus.

As shown in fig. 3, CRISPR/Cas 9-based amyB gene deletion plasmids pCC010, pCC019, and pCC022 are not as functional in bacillus licheniformis as all analyzed cells carrying the wild-type amyB locus.

Other promoter variants are functional in bacillus licheniformis, driving Cas9 expression. In particular, the gene deletion plasmids pCC014, pCC016 and pCC025, which have promoter variants PV4-5, PV4-7 and PV8-7, respectively, exhibited the highest gene deletion efficiency of more than 60%.

The correct single clone was streaked onto fresh LB agar plates without antibiotic followed by a second incubation at 48 ℃ overnight for plasmid elimination. The final clones were again analyzed by colony PCR for successful deletion of the amyB gene and plasmid loss by plating on LB agar plates containing 20. Mu.g/ml kanamycin. The resulting B.licheniformis strain with the plasmid deletion (kanamycin sensitivity) eliminated and the amyB gene deleted was designated B.licheniformis P310.

Example 2: gene deletion and gene mutation in Bacillus licheniformis using promoters PV4-5 and PV8-7

Electrocompetent bacillus licheniformis P308 cells were prepared as described above and transformed with 1 μ g of hag deletion plasmids, pCC029 and pCC030, each isolated from escherichia coli INV110 cells (Life technologies) with promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046), respectively, followed by plating on LB agar plates containing 20 μ g/ml kanamycin and incubation at 37 ℃ overnight.

The following day, 20 clones of each transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 087 and SEQ ID 088 to analyze successful deletion of the hag gene based on CRISPR/Cas9 and further transferred to fresh LB agar plates without antibiotics followed by overnight incubation at 48 ℃ for plasmid elimination.

The hag gene deletion efficiency of each CRISPR/Cas 9-based deletion plasmid was calculated as the ratio of successful gene deletions in percent relative to the total number of analyzed clones based on the appearance of the expected smaller specific PCR amplicon compared to the larger specific PCR amplicon of the wild-type hag locus. Three experiments were performed for each hag gene deletion plasmid. As shown in fig. 4A, CRISPR/Cas 9-based gene deletion efficiencies for plasmids pCC029 and pCC030 were 95% and 100%, respectively.

To analyze the efficiency of point mutation introduction, bacillus licheniformis P308 cells were transformed with two degU mutant plasmids, pCC031 and pCC032, which again differed in the promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046) driving constitutive expression of Cas9, as described for deletion of the hag gene. Transformed B.licheniformis cells were plated on LB agar plates containing 20. Mu.g/ml kanamycin, followed by overnight incubation at 30 ℃. The mutation efficiency for introducing the H12L degU mutation was calculated as the ratio of the successfully mutated degU gene in percent based on the occurrence of degU-specific PCR-amplicons which can be cleaved with the restriction endonuclease PstI when compared to the native degU-specific PCR-amplicon of the wild-type degU locus with the oligonucleotides SEQ ID 089 and SEQ ID 090, relative to the total of 20 analyzed clones. Three experiments were performed for each degU gene deletion plasmid. As shown in fig. 4B, the CRISPR/Cas 9-based mutation efficiencies of plasmids pCC031 and pCC032 are 19% and 24%, respectively.

Example 3: gene deletion in Bacillus subtilis Using promoters PV4-5 and PV8-7

Electrocompetent B.subtilis ATCC6051a cells were prepared as above and transformed with 1. Mu.g each of amyE deletion plasmids pCC033 and pCC034 having promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046), respectively, isolated from E.coli DH10B cells, then plated on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃.

The following day, 20 clones of each transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 091 and SEQ ID 092 to analyze successful deletion of the amyE gene based on CRISPR/Cas9 and further transferred to fresh LB agar plates without antibiotics followed by overnight incubation at 48 ℃ for plasmid elimination.

The amyE gene deletion efficiency of each CRISPR/Cas 9-based deletion plasmid was calculated as the ratio of successful gene deletions in percent relative to the total number of analyzed clones based on the appearance of the expected smaller specific PCR amplicon compared to the larger specific PCR amplicon of the wild-type amyE locus. Three experiments were performed for each hag gene deletion plasmid. As shown in fig. 5A, the CRISPR/Cas 9-based amyE gene deletion efficiencies of plasmids pCC033 and pCC034 inside bacillus subtilis were 97% and 100%, respectively.

Plasmids pCC035 and pCC 036-dependent promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046) were analyzed for gene deletion efficiency for deletion of the Bacillus subtilis aprE gene, similar to the method described for deletion of the amyE gene, however cells were incubated on LB agar plates containing 20. Mu.g/ml kanamycin, followed by transformation at 30 ℃ overnight. Gene deletions were again analyzed by colony PCR with oligonucleotides SED ID 093 and SEQ ID 094 and gene deletion efficiency was calculated as described above for three independent transformation reactions. As shown in fig. 5B, the CRISPR/Cas 9-based aprE gene deletion efficiencies of plasmids pCC035 and pCC036 inside bacillus subtilis were 32% and 47%, respectively.

Example 4: gene deletions in B.licheniformis using the promoters PV4-5 and PV8-7 and different spacers

Electrocompetent bacillus licheniformis Bli #005 cells were prepared as described above and transformed with 1 μ g of each of the vpr deleted plasmids pCC037, pCC038 and pCC039 with the promoter PV4-5 (SEQ ID 037) and different vpr unique spacers (SEQ ID 066-068), respectively, isolated from large intestine rod Ec #098 cells, then plated on LB agar plates containing 20 μ g/ml kanamycin and incubated overnight at 37 ℃.

The following day, 20 clones of each transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 095 and SEQ ID 096 to analyze successful CRISPR/Cas 9-based deletion of the vpr gene and further transferred to fresh LB agar plates without antibiotics followed by overnight incubation at 48 ℃ for plasmid elimination.

The vpr gene deletion efficiency of each CRISPR/Cas 9-based deletion plasmid was calculated as the ratio of successful gene deletions in percent relative to the total number of analyzed clones based on the appearance of the expected smaller specific PCR amplicon compared to the larger specific PCR amplicon of the wild-type vpr locus. As shown in fig. 6A, CRISPR/Cas 9-based vpr gene deletion efficiencies for plasmids pCC037, pCC038, and pCC039 were 100%, and 84%, respectively.

The gene deletion efficiency of the B.licheniformis epr gene deletion was determined for plasmids pCC040, pCC041 and pCC042 having the promoter PV4-5 (SEQ ID 037) and a different epr-specific spacer sequence (SEQ ID 070-072) as described for the vpr gene, however, oligonucleotides SEQ ID 097 and SEQ ID 098 were used for colony PCR based gene deletion analysis. As shown in fig. 6B, CRISPR/Cas 9-based epr gene deletion efficiencies for plasmids pCC040, pCC041 and pCC042 are 87.5%, 100% and 100%, respectively.

Example 5: gene integration in B.licheniformis using the promoters PV4-5 and PV8-7

Electrocompetent Bacillus licheniformis Bli #005 cells were prepared as described above and transformed with 1. Mu.g of the gene integrating plasmid pCC043 with the promoter PV4-5 (SEQ ID 037) isolated from E.coli Ec #098 cells, followed by plating on LB agar plates containing 20. Mu.g/ml kanamycin and incubation overnight at 37 ℃.

The following day, 20 clones of the transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 009 and SEQ ID010 to analyze the successful integration of the CRISPR/Cas9 based PaprE-GFPmut2 expression cassette to replace the bacillus licheniformis amyB gene and further transferred to fresh LB agar plates without antibiotics followed by overnight incubation at 48 ℃ for plasmid elimination.

The gene integration efficiency of the CRISPR/Cas 9-based gene integration plasmid pCC043 was calculated as the ratio of successful gene integration in percent relative to the total number of analyzed clones based on the appearance of the expected specific PCR amplicon compared to the more specific PCR amplicon of the wild type amyB locus. The experiment was performed twice. As shown in fig. 7, the efficiency of gene integration of plasmid pCC043 into Bli #005 based on CRISPR/Cas9 was 67%.

The gene integration efficiency of the PaprE-GFPmut2 expression cassette using plasmid pCC043 was determined in a similar manner to bacillus licheniformis P308 strain, showing an average gene integration efficiency of 72% in two independent transformation reactions, as described in figure 7.

Example 6: gene deletion in Bacillus pumilus using promoter PV4-5

Electrocompetent bacillus pumilus DSM14395 cells were prepared as described above and transformed with 1 μ g of sporulation gene deletion plasmids pCC044 (sigE), pCC045 (sigF) and pCC046 (spoIIE) with promoter PV4-5 (SEQ ID 037) driving expression of Cas9 endonuclease. Plasmid DNA was isolated from E.coli DH10B cells and methylated in vitro as described above, followed by transformation. Transformed B.pumilus cells were plated on LB agar plates containing 20. Mu.g/ml kanamycin and incubated overnight at 37 ℃.

The next day, 20 clones of each transformation reaction were subjected to colony PCR, with oligonucleotides SEQ ID099 and SEQ ID 100 for analysis of sigE deletions, oligonucleotides SEQ ID 101and SEQ ID 102 for sigF deletions and oligonucleotides SEQ ID 103and SEQ ID 104 for analysis of spolie deletions. The single colonies were further transferred to fresh LB agar plates without antibiotics and subsequently incubated overnight at 48 ℃ for plasmid elimination.

The gene deletion efficiency of plasmids pCC044, pCC045 and pCC046 in Bacillus pumilus was calculated as the ratio of successful gene deletions in percent relative to the total number of analyzed clones based on the appearance of the expected smaller specific PCR amplicon compared to the larger specific PCR amplicon of the wild type locus. As shown in fig. 8, the CRISPR/Cas 9-based gene deletion efficiencies of plasmids pCC044, pCC045 and pCC046 inside bacillus pumilus were 43%, 56% and 50%, respectively.

Sequence listing

<110> Pasteur European Co

<120> method for producing constitutive bacterial promoters conferring low to moderate expression

<130> 191606WO01

<160> 104

<170> according to Wipo Std 25

<210> 1

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of pE194

<400> 1

tatatacagc tggattcaca aaaaataggc ac 32

<210> 2

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of pE194

<400> 2

tatatacagc tggattatgt cttttgcgca gtc 33

<210> 3

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification functional mRFP cassette

<400> 3

tatatggatc cgtaatcagg gtatcgaggc 30

<210> 4

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification functional mRFP cassette

<400> 4

tatatggatc cctcattagg cgggctacta a 31

<210> 5

<211> 1345

<212> DNA

<213> Artificial sequence

<220>

<223> functional fragment: T2A mRFP box

<400> 5

ggaatagatc tggccaacga ggcctcgagg atccgatatc atgcatggcg cgccaccctg 60

agacgaccct gatgcaggtg accctgagac cttcgcgccc agctgtctag ggcggcggat 120

ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt 180

tcgactgagc ctttcgtttt atttgatgcc tttaattaag taccttgtcg gataaagctg 240

tgttatatta tgtcttggtg ttaaatacac acgcttaacg atttatgcag agggtgctgc 300

aggcggcagt tctgtacaaa aatgacctaa gcggaggaaa aaaaccatta tattaggagg 360

aaataacatg gcctcttcag aggatgttat taaagaattt atgcggttta aggtgaggat 420

ggaaggctcg gtgaacggac atgagttcga aattgaggga gaaggtgaag gccgccctta 480

tgaaggtact cagacagcga aattgaaagt cacgaaaggc ggaccgctgc cgtttgcttg 540

ggacattctc tcacctcaat ttcaatatgg ctcaaaagcc tacgtaaaac acccggctga 600

catccctgat tacttaaagc tatccttccc ggagggcttt aaatgggaac gagttatgaa 660

ttttgaggac ggcggcgtcg ttactgtcac acaggattct tcccttcagg atggcgaatt 720

tatttacaaa gtaaaacttc gtggaactaa cttcccaagt gatggtcccg tgatgcaaaa 780

aaaaacaatg ggatgggaag catctacgga acgtatgtat ccggaggatg gagccttaaa 840

gggtgaaatc aaaatgcgcc tgaaacttaa agatggcgga cactatgacg cggaagttaa 900

aacaacatat atggctaaaa aaccagtcca actgccggga gcatataaga cggatataaa 960

gttggacatt accagccata atgaagatta cacgattgtg gaacagtatg agagagcaga 1020

gggcagacat agcacaggcg cgtaagaatt aatgaaaaat aagcggcagc ctgcttttcc 1080

atgcgggctg ccgcttatcg ggttattgtc gtgactggga aaaccctggc gactagtctt 1140

ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg 1200

ctcggttgcc gccgggcgtt ttttattggt gagaatccag gggtccccaa taattacgat 1260

ttggtctcac tcacacctgc tcgtctcact caatttaaat ggcggccgcg gatcctcgac 1320

gggccaataa ggccagatct ggatt 1345

<210> 6

<211> 1012

<212> DNA

<213> Artificial sequence

<220>

<223> homology region: fusion of the 5 and 3 primer regions of the amyB gene to the flanking XmaI site

<400> 6

cccgggataa tgccgtcgca ctggccgata ttgagagatt tccttgtgac aagctgcaaa 60

gcataatgat gacggtccag ctcgcggctg attcccgtta acagattcat ataataaggt 120

tctgttgtat ccatttcttc cagtatgagc agcttgacga cctgtgttct gttttgaacg 180

agcgctcttg ctgcatagtt cggtatataa ttgagctcct tcattgcgga atgaacaagc 240

tttttcaatt catccgtcac agtctcagga tgattgatca cccgcgatac cgtcattttc 300

gacacatttg ctttctttgc tacatcagat aacgttgcca tttcatcccc gccttaccta 360

tgcgattcaa actgtcagca agtccttcct gagggctgat gacactttgt taaaattaat 420

tataaaatgt aatcaaagaa atttataaga cgggcaaaat aaaaaaacgg atttccttca 480

ggaaatccgt cctctctgct cttctagatt ctcctcccct ttcaatgtga aacatatgat 540

attgtataaa tattccgaat ttttaacaaa taccattttc cctatatttt cttccaaaag 600

aaaagcgccg atatggcgct ttctactcat ttattcaata gcctctctgc ttcttcactt 660

cttcaagctg agatacagtt accaattgat agccttttgc tttcagcttt ttaataatct 720

cttcagcagc atctgcggac gttgcataaa tatcgtgcat taagacgatt tttccgtctc 780

ccgcatggct catgacatga ttgacaatct tttgcttatt tttgtacttc caatcttccg 840

gatcaacatc ccacaatgaa accttcagat tggaaagcga gcggacggaa tcattgatcc 900

cgccgtatgg aggacgcaag tgtacaggca ggtgtccgct gattttttcg atcatttctt 960

gcgtgtcgtt aatctcctga tacgcttttt cgtttgacag ccttgtcccg gg 1012

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: targeting the protospacer target sequence amyB Gene

with 4nt 5-prime extension

<400> 7

tacgtcgcag cagaaattaa gaga 24

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: protoseger target sequence for amyB gene with 4nt 5-primer extension

<400> 8

aaactctctt aatttctgct gcga 24

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of amyB genomic regions

<400> 9

ttgcccgaat acaacgacag 20

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of amyB genomic regions

<400> 10

caacaacagg ctgtctgacg g 21

<210> 11

<211> 2143

<212> DNA

<213> Bacillus licheniforms

<220>

<223> P300 DSM641 RMS: RMS zone and flanking zone

<400> 11

aacttctata aatgtaacga tacgatttat tgtttcatta aagtcttcct ttatttcatg 60

ttcccatatt cttttaatgt tccaatcctt ttcctcgtaa tatttattaa cttccttatc 120

tcttttttta tttctttcga gttttttctc ccaatattcc gtattacttt ttggtatatt 180

cccgtgtttt tcacacgcat gccagaaaca agaatcaatg aatatgacta ttttatattt 240

ctgtattact atatctggac taccgtataa tttcttaaca ttttttcgga atcttattcc 300

acggtgccat agttctttag taaccttatc ttctaatttt gaacgagatt tgattgcctg 360

catgtttttt cttctttgtt cttttgaaac cgtgtcagtc atagaagagt cctccaaagc 420

cacaataatt gtattctata aacgaggaag caagccctca agcttacccc ctcttagttc 480

cttttttgcc tacttattta tttgttttca ttttcaaatt catcataaga acccatttca 540

caaaaatcaa tggagtaatg ttgttcgctg tgtttataaa caattactga gtcaatattt 600

agtctttcca gcatttcata ggttagaagt tctttttcct ctaagttcat aacttgtctt 660

agtagccatt ctccaagagc actatttgga ttagacataa gtgctttact attgtcttgg 720

cataccttgg ctgataaaag cgatttgtct ggcaagcgta actgaaaagg tttatcacga 780

gctgggaaaa atgttgggaa tacattatga atccattttg gaattggtat ataaatctcg 840

ttagggtttc gtggtcgacc taaagcattc cattggttta gaccgctttt ttctggtaca 900

tgacgctttg agccacggtc tgaaaagagt ggaagaataa cgtgctcaag gttttcaaaa 960

ggattgacag ttggtgctgg aatttttgga atttcaaagc caaatagttt agccaattca 1020

tgataaggat tttctaagat ttcaacatta atttcttcaa taggtttatc agtgataaaa 1080

cgcttataaa gggtgctctt agtgacatta aagctgtatt cgtgtagacc gtcttcaaag 1140

gtgattgtat ttctgttgtt acttactttc acatttgtaa ttgaggagat ttcaaccaag 1200

tccattggct cttcaaaaat aagaattttc cctggctttc ttgttacaca gtggtatatc 1260

attgaatcaa taccatatgt tcttttagta aattcaattc tctcgttacg gagagaagca 1320

accgtgttta ttagctcttt tggagatttc ccacgataca agtctgagtc tttattgaat 1380

tcagctactt tttgaagagt atgaccatta ccatgaagaa aagtcttaat accaattccg 1440

acacgattta atgaagcgtc agcagaacag tctgacctcc ccaagttttc agctccaaat 1500

gcttcacaaa aagcattttc cacattcctt gagaccaaat aaggcgagtc actttcagag 1560

aacaaattgg atagcgaacc agttgagcgg agcatttgtt tgtatgtagt gcagttgatg 1620

gctggttgat tagtatagaa cattattttt cctcctcttt tatgcttgtc atttcttctt 1680

tcagacccaa aaggtagtca gctgatacgt tcaatgtttc agctattctt ttgaaagtgt 1740

ccaatgatgg agttctattt tcactttcat atagtgacca agtgcttcta gtgaccccga 1800

ctttttcagc gatttggctg ggtaataacc tacgagcttc tcttgcattt tgaatacgat 1860

ttccaaggaa aggtatcatt tttgcacctc caagatttgt tgttttcaga gtatcaccag 1920

aacccccgaa aatagtccaa agttagctaa cagcaaacaa ataaaaataa ataagttgtt 1980

tactcttagc aaacttgtta ctaaaatttg ataaagttat tcatttaatc cagctcttat 2040

gctaaaattg cattagcgga caagcttaat gtttgcaagg aggtataatt ttgacttatc 2100

gagtaggtag tatgtttgct gggataggtg gaacttgttt agg 2143

<210> 12

<211> 1146

<212> DNA

<213> Bacillus licheniformis

<220>

<223> coding region: restriction enzyme P300 DSM641

<400> 12

ttatttgttt tcattttcaa attcatcata agaacccatt tcacaaaaat caatggagta 60

atgttgttcg ctgtgtttat aaacaattac tgagtcaata tttagtcttt ccagcatttc 120

ataggttaga agttcttttt cctctaagtt cataacttgt cttagtagcc attctccaag 180

agcactattt ggattagaca taagtgcttt actattgtct tggcatacct tggctgataa 240

aagcgatttg tctggcaagc gtaactgaaa aggtttatca cgagctggga aaaatgttgg 300

gaatacatta tgaatccatt ttggaattgg tatataaatc tcgttagggt ttcgtggtcg 360

acctaaagca ttccattggt ttagaccgct tttttctggt acatgacgct ttgagccacg 420

gtctgaaaag agtggaagaa taacgtgctc aaggttttca aaaggattga cagttggtgc 480

tggaattttt ggaatttcaa agccaaatag tttagccaat tcatgataag gattttctaa 540

gatttcaaca ttaatttctt caataggttt atcagtgata aaacgcttat aaagggtgct 600

cttagtgaca ttaaagctgt attcgtgtag accgtcttca aaggtgattg tatttctgtt 660

gttacttact ttcacatttg taattgagga gatttcaacc aagtccattg gctcttcaaa 720

aataagaatt ttccctggct ttcttgttac acagtggtat atcattgaat caataccata 780

tgttctttta gtaaattcaa ttctctcgtt acggagagaa gcaaccgtgt ttattagctc 840

ttttggagat ttcccacgat acaagtctga gtctttattg aattcagcta ctttttgaag 900

agtatgacca ttaccatgaa gaaaagtctt aataccaatt ccgacacgat ttaatgaagc 960

gtcagcagaa cagtctgacc tccccaagtt ttcagctcca aatgcttcac aaaaagcatt 1020

ttccacattc cttgagacca aataaggcga gtcactttca gagaacaaat tggatagcga 1080

accagttgag cggagcattt gtttgtatgt agtgcagttg atggctggtt gattagtata 1140

gaacat 1146

<210> 13

<211> 1022

<212> DNA

<213> Artificial sequence

<220>

<223> homology region: fusion of the 5-primer region and the 3-primer region of the restriction enzyme gene to the flanking BsaI site

<400> 13

ggtctcgacc caacttctat aaatgtaacg atacgattta ttgtttcatt aaagtcttcc 60

tttatttcat gttcccatat tcttttaatg ttccaatcct tttcctcgta atatttatta 120

acttccttat ctcttttttt atttctttcg agttttttct cccaatattc cgtattactt 180

tttggtatat tcccgtgttt ttcacacgca tgccagaaac aagaatcaat gaatatgact 240

attttatatt tctgtattac tatatctgga ctaccgtata atttcttaac attttttcgg 300

aatcttattc cacggtgcca tagttcttta gtaaccttat cttctaattt tgaacgagat 360

ttgattgcct gcatgttttt tcttctttgt tcttttgaaa ccgtgtcagt catagaagag 420

tcctccaaag ccacaataat tgtattctat aaacgaggaa gcaagccctc aagcttaccc 480

cctcttagtt ccttttttgc ctacttattt atatttttcc tcctctttta tgcttgtcat 540

ttcttctttc agacccaaaa ggtagtcagc tgatacgttc aatgtttcag ctattctttt 600

gaaagtgtcc aatgatggag ttctattttc actttcatat agtgaccaag tgcttctagt 660

gaccccgact ttttcagcga tttggctggg taataaccta cgagcttctc ttgcattttg 720

aatacgattt ccaaggaaag gtatcatttt tgcacctcca agatttgttg ttttcagagt 780

atcaccagaa cccccgaaaa tagtccaaag ttagctaaca gcaaacaaat aaaaataaat 840

aagttgttta ctcttagcaa acttgttact aaaatttgat aaagttattc atttaatcca 900

gctcttatgc taaaattgca ttagcggaca agcttaatgt ttgcaaggag gtataatttt 960

gacttatcga gtaggtagta tgtttgctgg gataggtgga acttgtttag gctcaggaga 1020

cc 1022

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of restriction enzyme genomic regions

<400> 14

gacaatcccc ttttactgac c 21

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of restriction enzyme genomic regions

<400> 15

ctatttcatt tgcccagaca atcc 24

<210> 16

<211> 1363

<212> DNA

<213> Artificial sequence

<220>

<223> homology region: the 5-primer and 3-primer regions of the pga gene were fused to flanking BsaI sites

<400> 16

ggtctcgacc cgaacactga aattttagac cgggctggga tatcgagaca tatatagggg 60

cgtttaatgg cgaatacagt aacaatgaga atagtaagaa aaattaaaaa tgttaaagtt 120

tgatgaatta tcattgaaaa aaattaatgg ctttttaaat cctaggattt taacctaaaa 180

tctgaagaaa taaggtggat cgaacgactc acaaaatatt tggatttgtc aatgaatccc 240

gctttatgct aaaagagatt ttcatttttt gatagatggt ctgattgtca taggacggat 300

ttgttttgaa gagggaacat tggtgacttt ttaacctgtt cgaaaagagc gaaaatacta 360

aaagaaaaga gacatcccgg ctgacagccc atttaaaggg gattgcggcc gggggaaaaa 420

agagatcctg aatccatcct tcaacctttc atctgaaata gggagaaaag tacaaaaatc 480

ataatgtcga attttgaaag cgcatactta aaacgctgac aaaaatctga taggaattaa 540

gaactttcga tttccaaaaa tatcaataaa aagataggca ttaatgactc gggcgaggtg 600

atctttgtca cggaaaattt cgtcgtcttc tgttacataa tgccgattgt gatttcatag 660

tgaaccctga tcccggttat aaaagacctg tgaaaagcgg ccggtttgaa agggaaacac 720

gacaattttc ttaaccggtc agtgtataaa gttttataga aaatcaggag gatatataca 780

tggttttggg gttcatgttt attgtattct tttgaaggga ataaaaactg acaaatttcg 840

actgaagcaa aatttgaaaa tgcatcacct taccaattcg ggatgggaac cgcacctcat 900

gttcatgacc tctttagaat atttcccttc atctttttaa tccgcgctta ggtgaaaaag 960

ctgatcatgc tgtgctgagc gtttcttctc gctatgacgc tgctgtacat gcaaaaaaag 1020

tcctttaaat atcccagttg aatgacgatg aaagaggaaa gaagaggagg aacagatcaa 1080

ttgataaaaa aagcggcaaa caaaaagttg gttttgtttt gtggaattgc ggtgctttgg 1140

atgtctttat ttttaacgaa tcataatgat gtacgcgccg atacgatcgg cgagaaaata 1200

gcggaaactg ccagacagct tgagggtgcg aaatacagct acggcggaga gaagccgaaa 1260

acggggtttg actcgtcagg ctttgtgcaa tatgtgtttc aatcgctcga tattacgctt 1320

ccgagaacgg taaaggaaca atcgactctt ggctcaggag acc 1363

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of pga genomic region

<400> 17

aaagccttct cctctctatt 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of pga genomic region

<400> 18

ttcttgaaaa agacaaggtc 20

<210> 19

<211> 1027

<212> DNA

<213> Artificial sequence

<220>

<223> homology region: fusion of the 5-primer region and the 3-primer region of the aprE gene to flanking BsaI sites

<400> 19

ggtctcgacc cgaagttctt ttttaacata taggtaaaac aatacgaaaa aaggcgccaa 60

gtattgaaga attgcagcag ccgcggcatt tcccttttcg attgaagcaa aaaacgtata 120

ttgaacagta agcattccaa aaatggaaaa tactaaaatc gaacaaatat ctgttttttt 180

cttccatatc tgacacacat gttgaaaacc gtttttcatt gaaacatata acaagagaat 240

gactcccgat gccagaagcc tgacagagac aagcgagccg gcttcaaccg ctcccctttc 300

aaatatgtac tgtgcagcgc ttcccgataa tccccacaat gaagcccctg caagcaccat 360

caatacgcct ttcacatgag ctgatttcat atctttcacc cgtttctgta tgcgatatat 420

tgcatatttt aatagatgat cgacaaggcc gcaacctcct tcggcaaaaa atgatctcat 480

aaaataaatg aatagtattt tcataaaatg agctcaataa catattctaa caaatagcat 540

atagaaaaag ctagtgtttt tagcactagc tttttcttca ttctgatgaa ggttgttcaa 600

tattttgaat ccgttccatg atcgtcggat ggccgtattt aaaaatcttg acgagaaacg 660

gcgggtttgc ctcgctcagc ccggcttttg agagctcttg aaacgtcgaa accgctgcat 720

cgctgttttg cgtcagttca atcgcatact ggtcagcagc tttttcctga tgcctcgaaa 780

ctgcgttcgt aaatggagac gacgcgaaag agatgacccc catcagcatc agaagaagcg 840

gaagtgcggc tagatcggat tttcctgcaa tatgaaggct tcttccatag cggccgatga 900

tccgcttgta cagcttgtcg atcacataaa agacagcaag ggataaaagc agatacccgc 960

caagtcctat gtaaacatgc ttcatcacat agtgccccat ttcgtgcgcc atgatgctca 1020

ggagacc 1027

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of aprE genomic regions

<400> 20

ccggttgtca ttgatccttt a 21

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of aprE genomic regions

<400> 21

atcctcctgc aaaaaccgta t 21

<210> 22

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of pBW742

<400> 22

caaatttaca aaagcgactc gtgagttttc gttccactga g 41

<210> 23

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of pBW742

<400> 23

gaacgttgct ctagagttaa gggattttgg tcatgg 36

<210> 24

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of the terminator region of pMutin2

<400> 24

gtggaacgaa aactcacgag tcgcttttgt aaatttgg 38

<210> 25

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of the terminator region of pMutin2

<400> 25

catgaccaaa atcccttaac tctagagcaa cgttcttgcc 40

<210> 26

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> functional fragment: terminator region of pMutin2

<400> 26

ggggatctct gcagtgagat ct 22

<210> 27

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of terminator region in pCC009

<400> 27

ggggatctct gcagtcggga agat 24

<210> 28

<211> 128

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 28

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttggagaa taaatgtgga gaaagattaa ctaataagga 120

ggacaaac 128

<210> 29

<211> 128

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 29

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttgaagaa taaatgtgga gaaagattaa ctaataagga 120

ggacaaac 128

<210> 30

<211> 101

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 30

aacacgagcc catttttgtc aaataaaatt taaccggtat caacgttaat aagacgttgt 60

caataaaatt attttgacaa aattttaata atccaaatga g 101

<210> 31

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 31

ctattgagta tttcttatcc atgtttgtcc tccttattag ttaatctttc tccacattta 60

ttctccaaca cgagcccatt tttgtca 87

<210> 32

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 32

gattaactaa taaggaggac aaacatggat aagaaatact caataggc 48

<210> 33

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 33

ctattgagta tttcttatcc atgtttgtcc tccttattag ttaatctttc tccacattta 60

ttcttcaaca cgagcccatt tttgtca 87

<210> 34

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 34

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa atgggctcgt gttggagaat aaatgtggag aaagattaac taataaggag 120

gacaaac 127

<210> 35

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 35

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttggagaa aatgtggaga aagattaact aataaggagg 120

acaaac 126

<210> 36

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 36

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttgggaat aaatgtggag aaagattaac taataaggag 120

gacaaac 127

<210> 37

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 37

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgaacaa aaatgggctc gtgttggaga ataaatgtgg agaaagatta actaataagg 120

aggacaaac 129

<210> 38

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 38

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttgagaat aaatgtggag aaagattaac taataaggag 120

gacaaac 127

<210> 39

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 39

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttggaaat aaatgtggag aaagattaac taataaggag 120

gacaaac 127

<210> 40

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 40

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aagggctcgt gttggagaat aaatgtggag aaagattaac taataaggag 120

gacaaac 127

<210> 41

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 41

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttgaagaa aaatgtggag aaagattaac taataaggag 120

gacaaac 127

<210> 42

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 42

aattttgtca aaataatttt attgacaacg tcttattaac cgttgatacc ggttaaattt 60

tatttgacaa aaatgggctc gtgttgaaga ataaatgtgg agaaagatta actaataagg 120

gaggacaaac 130

<210> 43

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 43

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttgaagaa taaatgtgga aaagattaac taataaggag 120

gacaaac 127

<210> 44

<211> 128

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 44

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccc ggttaaattt 60

tatttgacaa aaatgggctc gtgtgaagaa taaatgtgga gaaagattaa ctaataagga 120

ggacaaac 128

<210> 45

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 45

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa aatgggctcg tgttgagaat aaatgtggag aaagattaac taataaggag 120

gacaaac 127

<210> 46

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 46

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

atttgacaaa atgggctcgt gttgaagaat aaatgtggag aaagattaac taataaggag 120

gacaaac 127

<210> 47

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> promoter variants

<400> 47

aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60

attttgacaa aaatgggctc gtgttgaaga ataaatgtgg agaaagatta actaataagg 120

aggacaaac 129

<210> 48

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 48

gaagttttag atgccactct tatccatcaa tccatcactg 40

<210> 49

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 49

catctcaaat ttcgcattta ttccaatttc ctttttgcgt g 41

<210> 50

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 50

cacacgcaaa aaggaaattg gaataaatgc gaaatttgag 40

<210> 51

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 51

gaccagtgat ggattgatgg ataagagtgg catctaaaac 40

<210> 52

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 52

caagctatgc ttgctcaagc 20

<210> 53

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 53

ctgttggttc gcttgagcaa gcatagcttg gacggttcag cgtgttaagc 50

<210> 54

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 54

ggtggtggtc tctaccccga tcgaagagcc attcgag 37

<210> 55

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 55

ggtggtggtc tcttgagctt cctctgtccg attgtcc 37

<210> 56

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 56

tacggcaatc tctgaaaaaa tgag 24

<210> 57

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 57

aaacctcatt ttttcagaga ttgc 24

<210> 58

<211> 814

<212> DNA

<213> Artificial sequence

<220>

<223> homology region: mutant degU Gene homology region with flanking BsaI site

<400> 58

ggtctcgacc cgatttggga ttgataccga cgctcagaaa atacttgaac acgatcgaag 60

attatcatgg aaaagcaaag attcatttcc aatgcatcgg agaatccgaa gaaagaagaa 120

tagcaccgcg gtttgaggtt gcactattcc ggcttgcaca ggaagcggtg acaaacgcct 180

taaaacactc cgaatcaact gaaattcatg ttaaagtaga agtgacaaaa gattttgtga 240

cgctgattat caaagacaat ggaaacggct ttgacttaaa agaagtaaaa ggcaagaaga 300

acaaatcttt cggtctgcta ggtatgaaag aaagagtcga tttgctcgaa ggctcaatga 360

caatcgattc gaaaataggt cttgggacat ttatattgat taaagttcca ctgtctttgt 420

aaagataatt gtaaaataga gacaaaagac atattgacca taaaagcggt gtgtttaaca 480

atgagaatgg ggaggcgtag cttgtgacta aagtaaatat tgtaattatt gacgatctgc 540

agttattccg tgaaggtgtc aaacggattt tggatttcga gcctaccttt gaagtagtgg 600

ccgaaggaga cgacggagat gaagcggctc gcattgtcga gcactaccat cctgatgttg 660

ttatcatgga tattaatatg ccgaatgtga acggagtaga agcgacaaaa caactggtcg 720

acttgtatcc ggaatcaaag gttattattt tatccatcca tgatgacgaa aactatgtta 780

cacatgcatt aaaaacagga gccctcagga gacc 814

<210> 59

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 59

tacgggattt cgaacctacc tttg 24

<210> 60

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 60

aaaccaaagg taggttcgaa atcc 24

<210> 61

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 61

ggttgcggtc tcatacgtga agatcaggct atcactggt 39

<210> 62

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 62

caacgggtct cttgagttgg caggccgctg aatttc 36

<210> 63

<211> 1435

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA and homologous regions: the protospacer region aprE-sgRNA,

aprE gene homology region with flanking BsaI site

<400> 63

ggttgcggtc tcatacggaa acaaacccat accaggagtt ttagagctag aaatagcaag 60

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120

tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaagcttag 180

gcccagtcga aagactgggc ctttttaata cgactcacta tagggtcgac ggccaacgag 240

gcccattttc ttctgctatc aaaataacag actcgtgatt ttccaaacga gctttcaaaa 300

aagcctctgc cccttgcaaa tcggatgcct gtctataaaa ttcccgatat tggttaaaca 360

gcggcgcaat ggcggccgca tctgatgtct ttgcttggcg aatgttcatc ttatttcttc 420

ctccctctca ataatttttt cattctatcc cttttctgta aagtttattt ttcagaatac 480

ttttatcatc atgctttgaa aaaatatcac gataatatcc attgttctca cggaagcaca 540

cgcaggtcat ttgaacgaat tttttcgaca ggaatttgcc gggactcagg agcatttaac 600

ctaaaaaagc atgacatttc agcataatga acatttactc atgtctattt tcgttctttt 660

ctgtatgaaa atagttattt cgagtctcta cggaaatagc gagagatgat atacctaaat 720

agagataaaa tcatctcaaa aaaatgggtc tactaaaata ttattccatc tattacaata 780

aattcacaga atagtctttt aagtaagtct actctgaatt tttttaaaag gagagggtaa 840

agataatagt aaaaagaagc aggttcctcc atacctgctt ctttttattt gtcagcatcc 900

tgatgttccg gcgcattctc ttctttctcc gcatgttgaa tccgttccat gatcgacgga 960

tggctgcctc tgaaaatctt cacaagcacc ggaggatcaa cctggctcag ccccgtcacg 1020

gccaaatcct gaaacgtttt aacagcggct tctctgttct ctgtcaactc gatcccatac 1080

tggtcagcct tattctcctg ataacgcgag acagcattag aaaaaggcgt aaccgcaaag 1140

ctcaaaacag aaaacaaaag caataacagc ggaagtgccg caagatcatg ccgcccttct 1200

aaatgaaaca tgctgcgggt taggcgaacc gtccgcttgt aaagcttatc aatgacataa 1260

aatccggcga gcgacacgag caaatagcca gccagaccga tgtaaacgtg cttcatgaca 1320

taatggccca tttcgtggcc cataataaac agaatttctg aatcgtcaag tttgttcagc 1380

gtcgtatccc acaatacaat ccgtttattg gccccaattc tcaagagacc cgttg 1435

<210> 64

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: protopegestator target sequences for aprE gene

<400> 64

gaaacaaacc cataccagga 20

<210> 65

<211> 1241

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA and homologous regions: the protospacer region vpr-sgRNA,

vpr gene homology region with flanking BsaI site

<400> 65

ggttgcggtc tcatacgatc aggaacggaa cgagtcggtt ttagagctag aaatagcaag 60

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120

tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180

cagtcgatac ggatccacta gtctatctct tctctttttt ccgaaaagcc gcctgcctga 240

taagcatcgg cagcctgata tgtaccgccg gcgaacgccg tcaccttcat ctgattttct 300

ctgacaggca tcaccgcatt cagcagcacg gctgtccata attttaaaaa tgatatcaaa 360

cctttcatac cgatccctcc agtttcgttt tgataaaact agcaactcta ttaaactttc 420

ttgctctatc ttatcccagc aaaatgaaaa tgtttgtcac aatgtgtgtg caaaatgatt 480

ctagttttta gaagttttgt tgaaaactga aggaatcgca tgattcagcg gatacaaacc 540

atgaatgtaa cttactcaca gcttatccta aggataaaca catattaccc acaggatata 600

tccacatatc cacatactta ttcaatattt agtataagaa cgtatattcc ctacaatatc 660

tatacacaag tttattcact tatacacagt aaattgtgca taaatctaat gacaagcctt 720

gttgagaacc actcaacaag gcttttttat gttaaaatac ggataatgcg ttcaggagaa 780

gctccccttc tcttcaaaac gtgaaaaaag caatcggagg acatcgtgta tatgctttct 840

tttatcgtat tattcggctt atccttcatt attgtctgct ttatattttt cacgactttg 900

tacttcgccg tcaacctgca gaagcgcgag cccaagcctt ttcaaaaagc tgcggagcaa 960

accgtcgata ccatcatcct cattccgctc agctggctgt ttaccgcttt atacatatgc 1020

attctgttta ttcttttccc aatccgccat tttctcgatt tttttcagca aaaacgctaa 1080

attgactgat gaaacgcttc ggccagcagc cggtatgaat ccaatctgtc ttgaaaatcg 1140

tgggtgatcg tcaccgccat gatttcgtcc gttccgtaag cgccggccag ttcaagcagc 1200

tgttcctagc tcgagccatg gctcactcaa gagacccgtt g 1241

<210> 66

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: proto-spacer target sequence for the vpr gene

<400> 66

atcaggaacg gaacgagtcg 20

<210> 67

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Procompartmental region: proto-spacer target sequence for the vpr gene

<400> 67

gcttccgtat aatgagtatt 20

<210> 68

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: proto-spacer target sequence for the vpr gene

<400> 68

cacgatccga aaaacccgta 20

<210> 69

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA and homology regions: the protospacer region epr-sgRNA,

epr gene homology regions with flanking BsaI sites

<400> 69

ggttgcggtc tcatacgatt ccggtccagc gcttttagtt ttagagctag aaatagcaag 60

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120

tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180

ggcccagtcg atacgtaaag actgggcctt tttaatacga ctcactatag ggtcgacggc 240

caacgaggcc tcgaggatcc gatatcatgc atggcgcgcc accctgagac gaccctgatg 300

caggtgaccc ggatccacta gtccgatttg acatcgtgct gtttaaagga cctgacaaag 360

atatattcat taaaagggtg atcgggcttc cgggcgaaac cctcaggtat gaagatgatc 420

agctgtatat caacgaagaa aagatcaaag agccttatct ggacgactta aaggccgtca 480

ccgccggagg ggacttgaca ggggatttta cactgcagga agtgaccgga gaggagaagg 540

tgcctgaaaa cgagtacttc gtcctcgggg acaaccggat ccacagcttt gacagccgcc 600

atttcggctt tgtttcagaa cgggacatcg tcgggattgt gacggaaaga attgataaga 660

agtgattgga gagtacgggg gagagtaagc ggccgaccaa ggaatacgat tacgcaaatg 720

acgagcccga aatgtcaatt agtacaacag catcaataat gacgcatttg ctaaatatga 780

aaattaaaag gcccggatga ttccgggctt ttttccgtac taagcggcgt tcgctatata 840

tatcggagga tttttgaatt ttcaaagaga aagaaagctt atcttaaggt cgcttgtcat 900

gcacctttag tttttaaaac gttaaaaaca ctgttatatc aacatttgtg aagcttcctg 960

tttattcggg aagttaaatt gggtactcca agttagtttt aaaaaagagt cataaggcca 1020

gcttctatcg atgaatcatt tttaagcgac gccttttgtc taaaatgtat aatgttactt 1080

ttgtttttgt tgaaagtgaa caatgttatt gactggctta caacctacaa tttaattaaa 1140

taaaaaatag attaaaagaa gggagcgttc tcataccgtg gaaaaaacaa ttaaacatga 1200

ccccaattat tacaaaaaga taattattgc attgtgttta gggtgggtcg ctatttggat 1260

ttatcgtaca atacttacgc caatatatcc gcagattcaa gaatcattag ggaatattag 1320

tgctcaagag acccgttg 1338

<210> 70

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: protospacer target sequences for the epr gene

<400> 70

attccggtcc agcgctttta 20

<210> 71

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: protospacer target sequences for the epr gene

<400> 71

cgctttttca gctttggcaa 20

<210> 72

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: protospacer target sequences for the epr gene

<400> 72

gcaaacatgc cggtgacgtg 20

<210> 73

<211> 865

<212> DNA

<213> Artificial sequence

<220>

<223> functional fragment: T2A lacZ cassette

<400> 73

gcgatgttaa ggaccgtctc atctcacctg caaggtctca tctcttttac tccatctgga 60

tttgttcaga acgctcggtt gccgccgggc gttttttatc taaaactagt gtcgagggtc 120

ttcggtaccg cgatttacat atgctggcac gacaggtttc ccgactggaa agcgggcagt 180

gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt 240

atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 300

agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactctag aggatccccg 360

ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 420

cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 480

agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct 540

gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 600

cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 660

tgccgcgttt ataatgaaga ccctagcagg catcaaataa aacgaaaggc tcagtcgaaa 720

gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat 780

ccgccgccct agacagctgt cgctgagacg atcgctgaga cctcgcaagt tctcgccatc 840

gcaggtgaaa tctagatgta ttcgc 865

<210> 74

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification promoter PaprE

<400> 74

tatatgaaga ccttcgactc gggacctctt tccctcg 37

<210> 75

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification promoter PaprE

<400> 75

tatagaagac ttatatctca ctctcctcct ctttattcag a 41

<210> 76

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<223> functional fragment: GFPmut2 AF302837 with flanking BpiI restriction sites

<400> 76

attagaagac ctatatgagt aaaggagaag aacttttcac tggagttgtc ccaattcttg 60

ttgaattaga tggcgatgtt aatgggcaaa aattctctgt cagtggagag ggtgaaggtg 120

atgcaacata cggaaaactt acccttaaat ttatttgcac tactgggaag ctacctgttc 180

catggccaac acttgtcact actttcgcgt atggtcttca atgctttgcg agatacccag 240

atcatatgaa acagcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa 300

gaactatatt ttacaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag 360

gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca 420

ttcttggaca caaaatggaa tacaactata actcacataa tgtatacatc atggcagaca 480

aaccaaagaa tggaatcaaa gttaacttca aaattagaca caacattaaa gatggaagcg 540

ttcaattagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac 600

cagacaacca ttacctgtcc acacaatctg ccctttccaa agatcccaac gaaaagagag 660

atcacatgat ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac 720

tatacaaata atagcttgtc ttcatta 747

<210> 77

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of amyB genomic regions

<400> 77

tataggtctc aacccataat gccgtcgcac tgg 33

<210> 78

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of amyB genomic regions

<400> 78

ccttggtctc gtcgctagaa gagcagagag gacgg 35

<210> 79

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of amyB genomic regions

<400> 79

ttgagaggtc tcagagacat tttccctata ttttcttcc 39

<210> 80

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of amyB genomic regions

<400> 80

gtgaggtctc atgagaccat ccgttattga caagg 35

<210> 81

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: protosegenerate target sequences for sigE genes

<400> 81

cggtgaggaa aaaacccaaa 20

<210> 82

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA and homologous regions: the protospacer region epr-sgRNA,

sigE gene homology region with flanking BsaI site

<400> 82

ggttgcggtc tcatacgcgg tgaggaaaaa acccaaagtt ttagagctag aaatagcaag 60

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120

tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180

ggcccagtcg atacgtaaag actgggcctt tttaatacga ctcactatag ggtcgacggc 240

caacgaggcc tcgaggatcc gatatcatgc atggcgcgcc accctgagac gaccctgatg 300

caggtgaccc ggatccacta gtaagacagt atgatgaaca agtgcttgtg gaactacaca 360

ttcacggaga gacaattcgt ttaaagggac tcgtcgattc tggcaaccag ctgtatgatc 420

ctatgaccaa aacaccggtc atgatcgtcc aggccgacca tctaacggcc atttgcggag 480

aatcgtttat agaccttatg aaacagtctc atcctgttga agtcatgcaa aagatcgatg 540

atcaatttcc tcttcttgat cgattaagac ttgttccata tcgagcagtc ggtcatgatc 600

acggttttct actatgccta aaaccagata cagttgtcat ttattcaaag acgcatatga 660

ttcagccagc taagtgtttt gtaggattga gtctgagcgg cttatcggca gatcaggaat 720

ttcaatccat cattcatcca gatatgttag acgggaaaat catccagggg gtgtcgtagt 780

ttttggtgat gtcttttatc ttacgaggtc aacttgacat tttgaaaaat ttttttgaaa 840

agctctgtcc ctgtactgtc aaaggaaaca accttttttc tcatgattct cgtcatcgct 900

cgtgcatatt tttccaaccc aaggagatac tgaactttgt acaacagctc ctgtagggag 960

ggaaaaaagt gtccagaaat aaagtggaaa tctgcggagt cgacacctcc aagctgcctg 1020

ttctgaaaaa cgacgagatg agaaaattgt tcagacagct gcaagatgag ggtgacgata 1080

cagcaagaga aaagctagtc aatggcaatt tacggttggt tttaagtgtg attcagcgtt 1140

tcaacaacag aggcgagtat gtcgatgatc tctttcaagt aggctgtatc ggattaatga 1200

aatcaattga taattttgat ttaagccaca atgtcagatt ttcaacttat gcggttccta 1260

tgatcatagg agaaatccgt cgatacttgc gtgataataa tccgattcgg gtgtctcgct 1320

cactcaagag acccgttg 1338

<210> 83

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: protospacer target sequences for sigF genes

<400> 83

tgtcgtcctc gctcaagaag 20

<210> 84

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA and homologous regions: the protospacer region epr-sgRNA,

sigF gene homology region with flanking BsaI site

<400> 84

ggttgcggtc tcatacgtgt cgtcctcgct caagaaggtt ttagagctag aaatagcaag 60

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120

tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180

ggcccagtcg atacgtaaag actgggcctt tttaatacga ctcactatag ggtcgacggc 240

caacgaggcc tcgaggatcc gatatcatgc atggcgcgcc accctgagac gaccctgatg 300

caggtgaccc ggatccacta gtaaattatc cgtatggagc cttcagagca aacggcattg 360

caaacattgg gggtggcatc atgaggaatg aaatgaacct gaccttctct gccttaagtc 420

aaaatgaatc ctttgcgagg gtgacagtcg cggcgtttat cgcccagctt gatccgacat 480

tagatgaatt aactgaaatc aaaaccgttg tgtcagaggc ggtgacaaac tccattatcc 540

atggctatga tgggaatcca gatggcaagg tgcatattga agtcacactt gatgatcatg 600

ttgtgtacct gaccatccgt gacgaaggaa tgggtattac agatcttgag gaagcaagac 660

agccgctttt cacgacaaaa ccagacttag aacgctctgg catgggcttt accattatgg 720

agaattttat ggatgatgtc atgatagact catctccaga aatgggcaca accatccgtt 780

taacaaagca tctatcaaaa agcaaagcgc tttgtaatta aatagccaat tcggctggct 840

ttttttgtgt ggtaattacc ggtaaatgaa gttctctcgg tatgagaacc atttttcacc 900

acatactatt tttaacccat cgtataggaa gtgacttgga tggacggaca aatctttctt 960

cggctgcgcc atcgcattaa gaccggtaat gatcagctca tttatttaga agatatcgcc 1020

caaatcactg gtgatgagtt ggctgtgcaa aagcttagca agatgccgat atatcatgtc 1080

agtaaaaagg atcgtcacat tgccgttctt gatatcatgc atgtggtcaa aacgatcaaa 1140

aaaacatggc caaccatcga cattcaaact gtcggaggcg ctgaagccat tgttgaaatt 1200

gatacaggca aacgccagct ttctcccgta ttatttgtgt tcgtgtggct tttattattt 1260

gtcggagcgg cgcttgccat tatgaatttc cacgaggatg tcagtatgcg gctcgtccat 1320

atctcaagag acccgttg 1338

<210> 85

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Problocker: proto-spacer target sequence for spoIIE gene

<400> 85

cttgtagctg aacagctgat 20

<210> 86

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA and homologous regions: a protospacer region epr-sgRNA,

SpoIIE gene homology region with flanking BsaI site

<400> 86

ggttgcggtc tcatacgctt gtagctgaac agctgatgtt ttagagctag aaatagcaag 60

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120

tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180

ggcccagtcg atacgtaaag actgggcctt tttaatacga ctcactatag ggtcgacggc 240

caacgaggcc tcgaggatcc gatatcatgc atggcgcgcc accctgagac gaccctgatg 300

caggtgaccc ggatccacta gtcctgctgt ccgcaggtgc tttttttctt gacccacacg 360

acattttttg agatttcgtc atttaattta aaacttccta ttgacggaca agcgattcct 420

ttgtattata gatcttgtgc ttcttagcgc atttttatta tggcggtgta gctcagctgg 480

ctagagcgta cggttcatac ccgtgaggtc gggggttcga tcccctccgc cgctatcctt 540

ttgattagaa cataaaagca aggcccgttg gtcaagcggt taagacaccg ccctttcacg 600

gcggtaacac gggttcgaat cccgtacggg tcatcttcga aaacagcttt ctttaggaaa 660

gctgtttttt tgtgtcttca taaaattctg atgaaagaca tcgactttca agaaagtatg 720

cctctttgac gaataaagcg tcgaacgttt tatggaaacg acaacttctt ttgacaaaat 780

ttctttttca ccttcgctat aatgacaagc aacgaatatc agtgaaatat cgtataatat 840

gaatttcttc tggcgatgat ggggatataa agcattcagt acgatcccag gaggaatgaa 900

gatgcgaaaa ggtcacgtaa accaaatctt attgattaca gatggctgct caaatcacgg 960

ggaagatcca cttgcgattg cctcattggc aaaggaacaa gggattacag tcaatgttat 1020

tggcattatg gaggaaaaca gacacgacca tgaagcaatg aaagaagttg aagggattgc 1080

tctcgcaggt ggaggcatcc atcaagttgt ctacgtccag cagttatctc aaaccgtaca 1140

aatggttaca aaaaaagcga tgacacaaac cttgcaaggt gttgtgaata aagaattgca 1200

gcaaatactt ggcaaggaca ctgaaattga agagctgcca cctgataaac gcggggaagt 1260

gatggaagta gtcgatgagt taggagagac ggttcatctt caagtgcttg tgcttgttga 1320

tactcaagag acccgttg 1338

<210> 87

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of hag genomic region

<400> 87

tgatcttgat gaaacgacgg 20

<210> 88

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of hag genomic region

<400> 88

taatcctgat attctgatcg cc 22

<210> 89

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of the degU genomic region

<400> 89

atgatttaag gccgatggc 19

<210> 90

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of the degU genomic region

<400> 90

atccgccttc agctactact t 21

<210> 91

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of amyE genomic regions

<400> 91

ggtcatgaat aatctgcgta atagac 26

<210> 92

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of amyE genomic regions

<400> 92

gcgtgtacgt tttgaggcgc tgcgcc 26

<210> 93

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of Bacillus subtilis aprE genomic region

<400> 93

gagctggcag atgaagccaa tattcc 26

<210> 94

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<400> 94

gtacgcgcat gaggaacgac aaataag 27

<210> 95

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of vpr genomic regions

<400> 95

ctgtcaccca cttcccatta tgag 24

<210> 96

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of vpr genomic regions

<400> 96

gtgaccgaag gctttccatc attg 24

<210> 97

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of epr genomic regions

<400> 97

cttgtcatcg tcgtcgggat tcag 24

<210> 98

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of epr genomic regions

<400> 98

gtgccaatca caaatgtagc cagc 24

<210> 99

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of sigE genomic regions

<400> 99

gaggaggcat gatcggagtt cattc 25

<210> 100

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of sigE genomic regions

<400> 100

ctcctccgtc attatagatc ggttc 25

<210> 101

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of sigF genomic regions

<400> 101

gtgacgaatt atttggaaac agagg 25

<210> 102

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of sigF genomic regions

<400> 102

cgacataatg atcgagatcg agctg 25

<210> 103

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of spoIIE genomic regions

<400> 103

ctcaacaaca acaatcaaga ccgag 25

<210> 104

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide: PCR amplification of spoIIE genomic regions

<400> 104

gatagtgaat cgaatcgagg cgtcc 25

Claims

1. A method for the production of one or more synthetic regulatory nucleic acid molecules conferring reduced constitutive expression as compared to a corresponding initial regulatory nucleic acid molecule in a bacterial cell, said method comprising the steps of

b. operably linking said initial regulatory nucleic acid molecule to a coding region encoding a protein heterologous to said initial regulatory nucleic acid molecule, and

c. introducing into a vector comprising an origin regulatory nucleic acid molecule operably linked to a coding region, said vector comprising an origin of replication conferring high copy number to said vector in a bacterial cell, wherein said construct confers high expression of said coding region, wherein high expression of said coding region in a bacterial cell stresses said bacterial cell resulting in reduced or eliminated growth, and

d. transforming said vector into a bacterial cell, and

e. cultivating said transformed bacterial cell to recover a single clone, and

f. isolating a single clone that exhibits a growth rate comparable to a corresponding bacterial strain not comprising said construct, and

g. isolating the construct from the clone; and

h. testing the synthetic regulatory nucleic acid molecule comprised in said construct for functional expression of a gene operably linked to said synthetic regulatory nucleic acid molecule; and optionally

i. Sequencing the corresponding regulatory nucleic acid molecules comprised in said construct, thereby identifying a synthetic regulatory nucleic acid molecule conferring reduced constitutive expression in a bacterial cell.

2. The method of claim 1, wherein the synthetic regulatory nucleic acid molecule confers reduced expression in a bacterial cell different from the cell in which the recombinant nucleic acid is produced.

3. The method of claim 1 or 2, wherein the synthetic regulatory nucleic acid molecule is active in cells of gram-positive and gram-negative bacteria.

4. The method of claim 3, wherein the synthetic regulatory nucleic acid molecule is active in a cell of the class Bacillus (Bacillus) or gamma-Proteobacteria (Gamma).

5. The method of claim 4, wherein the synthetic regulatory nucleic acid molecule is active in cells of the Bacillaceae family (Bacillaceae) and Enterobacteriaceae family (Enterobacteriaceae).

6. The method of claim 5, wherein the synthetic regulatory nucleic acid molecule is active in a cell of the genus Bacillus (Bacillus) or Escherichia (Escherichia).

7. The method of claim 6, wherein the synthetic regulatory nucleic acid molecule is active in a cell of the genus Bacillus.

8. The method of claim 7, wherein the synthetically modulated nucleic acid molecule has activity in Bacillus alkalophilus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), bacillus circulans (Bacillus circulans), bacillus clausii (Bacillus clausii), bacillus coagulans (Bacillus coagulans), bacillus firmus (Bacillus firmus), bacillus lautus (Bacillus lautus), bacillus lentus (Bacillus lentus), bacillus licheniformis (Bacillus licheniformis), bacillus megaterium (Bacillus megaterium), bacillus pumilus (Bacillus pumilus), bacillus stearothermophilus (Bacillus stearothermophilus), bacillus methylotrophicus (Bacillus methylotrophicus), bacillus cereus (Bacillus cereus), bacillus parahaemophilus (Bacillus subtilis), bacillus subtilis (Bacillus subtilis), or Bacillus subtilis.

9. The method of claim 8, wherein the synthetic regulatory nucleic acid molecule is active in cells of at least three different Bacillus species.

10. The method of claim 9, wherein the synthetic regulatory nucleic acid molecule is active in cells of at least two different bacillus species.

11. The method of claim 10, wherein the synthetic regulatory nucleic acid molecule is active in cells of at least one bacillus species.

12. The method of claims 7-11, wherein the bacillus species comprises at least one of bacillus subtilis, bacillus licheniformis, or bacillus pumilus.

13. The method of claim 12, wherein the synthetic regulatory nucleic acid molecule is active in a bacillus licheniformis cell.

14. The method according to claims 1 to 14, wherein the initial regulatory nucleic acid molecule conferring constitutive expression in a bacterial cell is selected from the group consisting of

a.SEQ ID NO. 28 and 29,

b. a nucleic acid molecule comprising at least 20 consecutive base pairs identical to the 20 consecutive base pairs of the sequence depicted in SEQ ID NO 28 or 29, and

c. a nucleic acid molecule having at least 90% identity over the entire length of the sequence depicted in SEQ ID NO 28 or 29, and

d. a nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of at least 20 consecutive base pairs of the nucleic acid molecule depicted in SEQ ID NO 28 or 29, and

e. the complement of any of the nucleic acid molecules as defined in a) to d).

15. Synthetic regulatory nucleic acid molecules, wherein the regulatory nucleic acid molecule is comprised in the group consisting of

a. A nucleic acid molecule having the sequence of SEQ ID NO 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

b. a nucleic acid molecule comprising at least 20 consecutive base pairs identical to 20 consecutive base pairs of the sequence set forth in SEQ ID NO 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

c. a nucleic acid molecule having at least 90% identity over the entire length of the sequence depicted in SEQ ID NO 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

d. a nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of at least 20 consecutive base pairs of the nucleic acid molecule of any one of SEQ ID NOs 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

e. the complement of any of the nucleic acid molecules as defined in a) to d),

wherein the sequences as defined in b) to e) differ from the corresponding starting nucleic acid molecules.

16. The synthetic regulatory nucleic acid molecule of claim 15, wherein the nucleic acid molecule is produced as described in any one of claims 1 to 14.

17. An expression construct comprising the synthetic regulatory nucleic acid molecule of claim 15 or 16.

18. A vector comprising the regulatory nucleic acid molecule of claim 15 or 16 or the expression construct of claim 17.

19. A microorganism comprising the regulatory nucleic acid molecule of claim 15 or 16 or the expression construct of claim 17 or the vector of claim 18.